Hypoxia-induced SZT2-AS1 is required for HIF-1 heterodimer formation and histone trimethylation in HCC cells under hypoxic microenvironment

Hypoxia-induced SZT2-AS1 is required for HIF-1 heterodimer formation and histone trimethylation in HCC cells under hypoxic microenvironment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hypoxia-induced SZT2-AS1 is required for HIF-1 heterodimer formation and histone trimethylation in HCC cells under hypoxic microenvironment Yufeng Wang, Runkun Liu, Yixian Guo, Guozhi Yin, Hang Tuo, Yifeng Zhu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4805397/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2024 Read the published version in Cell Death & Differentiation → Version 1 posted 9 You are reading this latest preprint version Abstract Hypoxic microenvironment plays a critical role in solid tumor growth, metastasis and angiogenesis. Hypoxia-inducible factors (HIFs), which are canonical transcription factors in response to hypoxia, are stabilized under hypoxia and they coordinate the process of hypoxia-induced gene expression leading to cancer progression. Increasing evidence has indicated that long noncoding RNAs (lncRNAs) which are closely associated with cancer play crucial roles in hypoxia-mediated HCC progression, while the mechanisms are largely unknown. Here, we identified a novel lncRNA SZT2-AS1 in HCC, which was induced by hypoxia in a HIF-1-dependent manner and promoted HCC growth, metastasis and angiogenesis. The clinical data indicated that SZT2-AS1 level was substantially upregulated in HCC and significantly associated with poor clinical outcomes, and acted as an independent prognostic predictor. Mechanistically, SZT2-AS1, in turn, recruited HIF-1α and HIF-1β to form the HIF-1 heterodimer. And SZT2-AS1 was required for the occupancy of HIF-1 to hypoxia response elements (HREs) and HIF target gene transcription. In addition, SZT2-AS1 was required for hypoxia-induced histone trimethylation (H3K4me3 and H3K36me3) at HREs. Through recruiting methyltransferase SMYD2, SZT2-AS1 promoted trimethylation modification of H3K4 and H3K36 in HCC cells. Taken together, our results uncovered a lncRNA-involved positive feedback mechanism under hypoxia and established the clinical value of SZT2-AS1 in prognosis and potential therapeutic strategy for HCC. Significance: LncRNA SZT2-AS1 involves in a positive feedback mechanism under hypoxia, which provides a therapeutic strategy for HCC. Biological sciences/Molecular biology/Epigenetics Biological sciences/Cancer/Cancer microenvironment hepatocellular carcinoma hypoxia SZT2-AS1 lncRNA SMYD2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Hepatocellular carcinoma (HCC) holds the third position for cancer-related mortality within the globe and the second deadliest cancer in China[ 1 ]. The characteristics of high malignancy, high mortality and high recurrence rate of hepatocellular carcinoma (HCC) make the current treatment still very challenging. Though surgical resection remains the most effective and curative strategy, the fact is that most HCC patients have already missed the best operation stage for surgical resection when they are diagnosed, while gene target therapy would be recommended as one of the treatments to the advanced HCC[ 2 ]. However, HCC progression is a complicated, multistep biological process, implicating multiple genes and biomolecules, which is a critical characteristic of all of the cancers. Therefore, in clinical practice, therapeutic effects of the current targeted drugs for HCC are still not very favorable[ 3 , 4 ]. And uncovering the molecular mechanisms of HCC will facilitate the improvement of early HCC diagnosis precision and the development of more efficient therapeutic targets. Hypoxic microenvironment is an intrinsic trait of solid tumors, and is strongly associated with an invasive phenotype and poor prognosis in many cancer types[ 5 ]. The transcription factors hypoxia-inducible factors (HIFs) are a kind of pivotal regulator of response to hypoxic stress, among of which HIF-1 is the most common one and in charge of at least hundreds of protein coding and non-coding RNA genes, which are involved in multiple aspects of tumourigenesis, including proliferation, angiogenesis, invasion, metastasis, glucose and energy metabolism, and cancer stem-like properties[ 6 , 7 ]. HIF is a heterodimer of an oxygen-regulated α subunit and a stably expressed β subunit, and the heterodimer formation is the cornerstone action for the occupancy of HIF to hypoxia response element (HRE) containing the core HIF binding sequence 5′-RCGTG-3′ (R = A or G) in target genes and the initiation of its transcription process[ 8 ]. In addition, a large body of evidence suggest that during the transcription process the co-activators of HIF are essential for HIF-mediated gene transcription, through binding to HIF-1α or HIF-1β or both to modulate the HIF protein stability, nuclear translocation, the occupancy of HIF to HRE, HIF activity or histone modifications[ 9 ]. It's worth noting that these co-activators not only include the proteins, such as FACT[ 10 ], NARF[ 11 ], NANOG[ 12 ], PADI4[ 13 ], some long non-coding RNAs like LncHIFCAR[ 14 ], DLEU1[ 15 ] are also identified as HIF-1 co-activators in recent years. Though lncRNAs are a cluster RNAs that have no ability to encode protein and are more than 200nt length, their diverse functional mechanism that are totally different from coding-genes make them the hot pint[ 16 ]. The mechanism of lncRNAs involved in included regulated gene transcription (in cis or trans), chromatin remodel, and mRNA splicing; produced endogenous small interfering RNAs (siRNAs) and microRNA precursors; change protein localization; regulate protein activity; and organized protein components[ 17 , 18 ].In our previous researches, plenty of lncRNAs associated with HCC progression have been identified. For instance, lncRNA CASC2 inhibits epithelial-mesenchymal transition (EMT) in HCC cells through the CASC2/miR-367/FBXW7 axis[ 19 ]. Additionally, our study investigated that MCM3AP-AS1 promotes HCC growth by targeting the miR-194-5p/FOXA1 axis[ 20 ]. Recently, especially with the booming development of high-throughput technology, more and more lncRNAs have been indicated to be related to tumor hypoxic microenvironment. For example, MAPKAPK5-AS1 expression is upregulated under hypoxia and promoted HCC progression via miR-154-5p/PLAGL2/HIF-1α signaling loop[ 21 ]. RUNX1-IT1 is restrained by hypoxia-induce histone deacetylase 3 to inhibits cancer stem-like properties and HCC cells proliferation[ 22 ]. KDM4A-AS1, which is induced by oxygen deficiency, promotes the growth of HCC and transition through the KDM4A-AS1/ KPNA2/HIF-1α signal circuit[ 23 ]. However, much more mechanism about hypoxia-related lncRNA remain further work to figure out. In this study, hypoxia-conditional RNA-seq in HCC cells was performed to scan and identify a cluster of hypoxia-regulated lncRNAs, where the novel hypoxia-induced lncRNA SZT2-AS1 was focused. Then, SZT2-AS1 was demonstrated to be induced by hypoxia in a HIF-1-dependent manner. In addition, the expression of SZT2-AS1 was upregulated in HCC and closely associated with clinical prognosis in HCC. Functionally, SZT2-AS1 promoted HCC growth, metastasis and angiogenesis, and mediated the hypoxia-induced HCC progression. Furthermore, in turn, SZT2-AS1 recruited HIF-1α and HIF-1β to form the HIF-1 heterodimer, meanwhile promoting the trimethylation modification of histones (H3K4me3 and H3K36me3) at HRE sites via recruiting SMYD2 to make chromatin decondensation, then increasing the occupancy of HIF-1 to HRE and HIF-1 transcriptional activity. Materials and methods Tissue samples Total 125 pairs of HCC tissue samples and adjacent non-tumor tissue samples, which were histopathologically confirmed, were collected from HCC patients stood hepatectomy in the First Affiliated Hospital of Xi’an Jiaotong University. Neither chemotherapy nor radiotherapy was administered before surgery to any of the patients. A temperature of -80°C was used to store all samples. Our study got approval from the Ethics Committees of the First Affiliated Hospital of Xi’an Jiaotong University, all patients provided informed consent. Cell culture The human normal liver cell line (MIHA), six cell lines (HepG2, Huh7, Hep3B, MHCC97H and SK-Hep-1) and human embryonic kidney (HEK) 293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All of the cells were maintained in incubator (37℃, 5% CO 2 ), and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Invitrogen, CA, USA). For hypoxia treatment, physical hypoxic condition (1%O 2 ) was generated by Forma Series II 3130 incubator (Thermo Scientific). Plasmids and cell transfection The small hairpin RNAs (shRNAs) targeting SZT2-AS1 (shSZT2-AS1#1, shSZT2-AS1#2) were obtained from GeneCreate Biological Engineering Co., Ltd. (Wuhan, China). HIF-1α, HIF-2α shRNAs and scrambled shRNA (shNTC) were purchased from GeneCopoeia (Guangzhou, China). The human HIF-1α or SMYD2 ORF cDNA clone (ov-HIF-1α, ov-SMYD2), and control empty vector were purchased from GeneCopoeia, Inc. All sequences were verified by DNA Sanger sequencing. Lentiviral production was achieved by transfecting target plasmids with psPAX2 packaging plasmid and pMD2.G envelope plasmids into HEK293T cells. Viral supernatant added to HCC cells with 8 µg/ml polybrene (Beyotime Biotech Inc., Shanghai, China), The transfected cells were treated with 3 µg/ml puromycin and 100 µg/ml Ampicillin to select knockdown and overexpressed single clone cells. Cell transfections were performed by using Lipofectamine 3000 reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) By using Trizol (Thermo Fisher Scientific) after completing the designated intervention, the RNA from HCC cells and tissues was extracted. A reverse transcription kit was used to reverse-transcribe total RNA into cDNA (Invitrogen, CA, USA). Real-time qPCR analysis was performed using SYBR Green Premix PCR Master Mix (Roche Diagnostics, Mannheim, Germany). Normalizing the relative expression level to 18S and the expression was calculated by 2 −ΔΔCt methods. The sequences of the primers used are listed in Supplementary Table 1. Western blot and immunoprecipitation (IP) assays RIPA buffer was used to isolate total protein from cells which supplemented with proteinase inhibitors and phosphatase inhibitors (Beyotime, Hangzhou, China). According to the manufacturer's instructions, BCA Protein assay kits (ZHHC Biotech Inc., Shaanxi, China) were used to determine protein concentration. Protein was separated by 10% or 8% or 15% concentration SDS-PAGE gels, then transferred to 0.22µm PVDF membranes (Millipore, Billerica, MA, USA). Following 3 hours of blocking by 10% nonfat milk, primary antibodies were used to incubate membranes at 4℃ temperature overnight. Afterwards, secondary antibodies conjugated to HRP were incubated on the membranes at room temperature for 1 hour. The blots were detected using enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA). For immunoprecipitation, equal amounts of WCLs (500 µg) were incubated with primary antibody HIF-1α (2 µg) in the presence of protein G-Sepharose beads (Amersham Biosciences) at 4°C overnight, and the immunoprecipitates were subjected to SDS-PAGE and immunoblot assays. Transwell migration and invasion assays As we previously reported[ 19 ], after completing the specified experimental processing, cell migration and invasion ability were investigated using Transwell migration and invasion assays following established protocols from prior research. Wound healing assay Wound healing assay was used to detect cell migration ability. After transfected HCC cells with different plasmid or virus, the experiment was conducted according to the protocols as we previously reported[ 24 ]. MTT assay and EdU assay Like MTT assay, specified transfected cells were added into 96-well plates at a density of 1 × 10 4 cells/ well. Then with 0, 24, 48, and 72h after seeding, 20µL MTT solution (Sigma, USA) was added to each well and continued incubate for 4h at 37℃. After removed the supernatants, added 100 µl DMSO to each well. Each absorbance was measured at 490 nm by a microplate reader (Bio-Rad, Richmond, CA). For EdU assay, Cell-Light™ EdU Apollo®567 (RiboBio Co., Ltd. Guangzhou, China) was used to evaluate of cell proliferation. Briefly, transfected HCC cells were cultured in 96-well plates. Then complete the experiment according to the protocol supply by manufacturer. The percentage of EdU positive cells was calculated using ImageJ software. EdU positive rate is calculated by counting at least five random fields. Tube formation assay The different treatment HCC cell culture medium was changed to serum-free DMEM medium for 48 h and then was collected, centrifuged and filtered to obtain tumor-conditioned medium (TCM). Prepare the dissolved Matrigel and precool the 24-well plate and pipets at -20℃. The precool 24-well plate was laid with 200µl Matrigel matrix (Corning Inc., Corning, NY, USA) incubated 37℃ for 30min. HUVEC (5×10 4 ) cells were added to each well with 200µl TCM which came from HCC cells and supplemented with 10% FPS, and then incubated at 37°C in 5% CO 2 for 8h. Pictures were taken under a bright-field microscope and the capillary tubes were quantified by counting branch number and total tube length with Image J. Dual-luciferase reporter assay The SZT2-AS1 promoter region sequence containing wild-type (WT) or mutated (MUT) sequences of hypoxia response elements (HREs) were embeded into pGL3-based vectors (Promega, USA). Luciferase reporter plasmid pGL3-based vectors expressing SZT2-AS1-WT or MUT were co-transfected into Hep3B and MHCC97H cells in 96-well plates with empty vector or pcDNA3.1/HIF-1α. After 24h, cells lysates were collected, the dual-luciferase reporter system kit (Beyotime, Shanghai, China) were used to measure Renilla and firefly luciferase activities on a microplate reader according to the manufacturer’s advices and protocols. Normalized luciferase activity according to The Renilla luciferase internal control. Bimolecular fluorescence complementation assay The pCMV-NRluc and pCMV-CRluc plasmids were constructed. The human bHLH-PAS domain of HIF-1α (amino acid residues 12–396) was prepared by PCR amplification and the PCR product was cloned downstream of the N-terminal segment (residues 1-229) of Rluc, in pCMV-NRluc-HIF-1α 12−396 . Similarly, the bHLH-PAS domain of HIF-1β (residues 11–510) was amplified and inserted upstream of the C-terminal segment (residues 230–311) of Rluc, in pCMV-HIF-1β 11−510 -CRluc. MHCC97H and Hep3B cells were seeded at 2x10 5 cells per well of a 24-well plate and incubated for 24h. Cells were co-transfected with 300 ng of NRluc-HIF-1α 12−396 , 300 ng of HIF-1β 11−510 -CRluc, and 80 ng of pGL2-promoter, using Fugene-6 (Roche) according to the manufacturer’s instructions. Following 7h incubation, cells were treated with indicated shNTC or shZST2-AS1 for 24 h. Cells were then lysed and analyzed for the ratio of Rluc/Fluc using the Dual Luciferase Assay System (Promega). RNA-seq analysis Hep3B cells were seeded into six-well plates for 24h, and then incubated in 20%O 2 or 1%O 2 for another 24h. Total RNA was isolated from the cells using TRIzol (Invitrogen) and treated with deoxyribonuclease (Qiagen). Library preparation and sequencing using the NovaSeq 5000 platform (Illumina) were performed. The FASTQ files were subjected to quality check and analyzed by Genialis Inc. ( https://www.genialis.com ). Differential expression results with a false discovery rate of 1.5 were used as acut off for further downstream analysis. RNA immunoprecipitation (RIP) RIP assay was conducted by utilizing the EZ-Magna-RIP kit (MilliporeSigma, Birlington, MA) according to the the manufacturer's instructions. Cells were lysed by lysis buffer which added with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA) and RNase inhibitors (Millipore Sigma). Then, the cell lysate was pre-washed with recombinant protein A/G agarose (Thermo Fisher Scientific) to reduce non-specific binding for 30 min at 4°C. One to twenty percent of the cell lysate were used as input. Then the specific antibody (anti-HIF-1α, anti-HIF-1β, anti-HIF-2α or IgG) and protein A/G magnetic beads were placed in equal amount of cell lysates for overnight at 4℃, negative control is used IgG. At the next day, RNA was eluted from the precipitated complex by using Trizol and transcribed into cDNA. RT-qPCR assay was performed to detect binding of RNA (SZT2-AS1) to proteins or antibody. The primary antibodies were listed in Supplementary Table 2. RNA pull-down assay The interaction between SZT2-AS1 and SMYD2 or HIF-1α or HIF-1β was predicted with RNA-Protein Interaction Prediction (RPISeq). Biotin labelled Sense and antisense of SZT2-AS1 RNA were in vitro transcribed with AmpliScribe T7-Flash Biotin-RNA Transcription Kit (Epicentre), treated with RNase-free DNase I and purified with a RNeasy Mini Kit (Qiagen). To establish the appropriate secondary structure, biotinylated SZT2-AS1 RNA supplied with RNA structure buffer (10mM Tris pH7, 0.1M KCl and 10 mM MgCl2) was first heated up to 90°C for 2 min, then incubated on ice for 2 min and last transferred to room temperature (RT) for 20 min. The RNA was then mixed with hypoxic Hep3B and MHCC97H cells extract or purified proteins and incubated at RT for 1 h, followed by incubating with Streptavidin Mag Sepharose (GE Healthcare) at RT for 1 h. After follow-up wash, extract the pull-down complexes were analysed by standard western blot technique. RNA Subcellular fraction To determine the cellular localization of SZT2-AS1, cytoplasmic and nuclear fractions were isolated and collected with the PARIS Kit (Life Technologies, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. Thereafter, collections from HCC cells both cytoplasm and nucleus were extracted out total RNA and cDNA was synthesized for the evaluation of SZT2-AS1. Briefly, we collected 1×10 7 cells and washed in PBS three times, and then 300µl Cell Fractionation Buffer resuspended cells and incubated at 4°C for 10 min. After 12000rpm centrifugation, aspiration of supernatant containing cytoplasmic components and 300µl Cell Disruption Buffer were resuspended collection of centrifugal precipitation containing nuclear fragments. The manufacturer’s instructions were used to extract RNA from the buffer containing cytoplasmic/nuclear fraction, following RT-PCR analysis of the levels of nuclear control transcript (U6), cytoplasmic control transcript (GAPDH) and SZT2-AS1. Protein isolation and analysis Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) was using to protein isolation according to the manufacturer’s instructions. Briefly, cells that have been treated in advance were harvested and dissociated in 200µl Reagent mixture containing 1 mM PMSF (Key Gen BioTech, Nanjing, China). The dissociated cell was incubated at 4°C for 15 min. Then, adding 10µl Reagent B and vortex the mixture for 5 s with 1 min ice bath, after that centrifugation at 16000 g, for 5 min. The supernatant we collected was cytoplasmic protein and 50µl nuclear protein extraction reagent containing 1 mM PMSF were further resuspend the precipitation. After being vortexed and 4°C in turn for 30 min, the mixture was centrifugated 5 min at 16000 g for 4°C, and the supernatant was stored as nuclear protein. The subcellular fractions were determined by using BCA protein assay kit, and then subjected to immunoblotting. Purification of GST-HIF-1α and In vitro RNA-binding assay Luria–Bertani medium with ampicillin (50µg/ml) used to culture Escherichia coli host BL21(DE3) harbouring the expression vector pGEX-6p-1-HIF-1a, and induced by 0.3 mM IPTG at 30°C for 16 h. Affinity purification of Recombinant protein by Pierce Glutathione Super Flow Agarose (Pierce) following the manufacturer’s instructions. SZT2-AS1 RNA was synthesized in vitro and added to RNA structural buffer (10 mM Tris pH 7, 0.1 M KCl and 10 mM MgCl 2 ), mixture first heated to 90°C for 2 min, follow incubated on ice for 2 min and then transfer to RT for 20 min to form the proper secondary RNA structure. GST fusion proteins on 20µl glutathione Sepharose beads were incubated with 2 mg SZT2-AS1 RNA synthesized in vitro which included in 50µl of RNA-binding buffer (0.1% NP-40, 100 mM KCl, 2 mM MgCl 2 , 50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol and ribonuclease inhibitor) for 30 min at 4°C. Follow up, the glutathione Sepharose beads were washed with RNA-binding buffer three times to remove non-attached RNAs. Trizol reagent were used to extract the RNA samples retained on the beads and detected expression by RT–qPCR. Calculate the relative retention value of the input RNA level. Chromatin immunoprecipitation (ChIP) MHCC97H and Hep3B cells were incubated at 20 or 1% O 2 for 16 hours and harvested for ChIP assay, cells cross-linked in 3.7% formaldehyde for 15 min, neutralized in 0.125M glycine for 5 min, and then used SDS lysis buffer to lysed HCC cells. Chromatin was sheared by sonication to an average length of 200–1000 bp, and salmon sperm DNA/protein A agarose slurry (Millipore) precleared cells lysates for 1 hour, protein A–agarose beads incubated with antibody against HIF-1α, HIF-2α, SMYD2, HIF-1β, H3K4me3, H3K36me3, H3 overnight at 4°C. Follow by serial washing of the agarose beads with low-salt, high-salt, and LiCl buffers, 1% SDS with 0.1 M NaHCO 3 elute DNA from beads, and reverse cross-links by addition of 0.2M NaCl. Purification of DNA by phenol-chloroform extraction and ethanol precipitation and amplified by RT-qPCR using primers listed in Supplementary Table 1. And antibodies were listed in Supplementary Table 2. RNA fluorescent in situ hybridization (FISH) FISH kit (RiboBio, Guangzhou, China) was used to detected the Subcellular localization of SZT2-AS1 according to the manufacturer’s procedure. In brief, MHCC97H and Hep3B cells were cultured on glass coverslips into 24-well plates. After 4% paraformaldehyde was utilized to fix HCC cells and washed with PBS, then subjected to permeabilization (0.5%Triton-X100 PBS). HCC cells were incubated with prehybridization solution and hybridized with hybridization solution, and then incubated with hybridization solution contain Cy3-labeled SZT2-AS1 oligonucleotide probe overnight. HCC cells nuclei were visualized with DAPI. All images were captured and recorded under a Zeiss fluorescence photomicroscope (Carl Zeiss AG). Animal experiments The growth and metastasis ability of cells in vivo were assessed by the orthotopic HCC model, subcutaneous xenograft model and lung metastasis model in mice. 4 weeks old male BALB/C nude mice purchased from the Centre of Laboratory Animals at The Medical College of Xi’an Jiao tong University and the mice were randomly grouped (n = 5 per group). All the animal experiments were approved by the Research Ethics Committee of Xi’an Jiao tong University. For the construction of orthotopic HCC model, 1×10 7 Hep3B-shSZT2-AS1 or MHCC97H-shSZT2-AS1 or control subclones were dissolved in 0.1 mL of DMEM culture medium. Mice were anesthetized with 3% pentobarbital sodium, and the liver was exposed by open surgery. The cells were injected into the liver of the nude mice, and the wound was sutured with 5 − 0 silk thread. 4 weeks later, the mice were sacrificed in accordance with ethical procedures to observe the tumor formation, and the liver tissues were used for H&E staining. In vivo subcutaneously tumor growth assay, 1×10 7 Hep3B-shSZT2-AS1 or MHCC97H-shSZT2-AS1 subclones and the same numbers corresponding control subclones were transplanted into the flank of 4-week-old BALB/c nude mice via subcutaneous injection. After injection, we measured tumor size calculated 0.5 × length × width × width every 3 days. The nude mice were killed after 21 days in different groups, and the tumor specimens were weighed, fixed and harvested for IHC experiments. In vivo lung metastasis model, we intravenously injected 1×10 6 cells into the lateral tail vein of nude mice. After 5 weeks killed the mice and collect the lung tissues. Thereafter, the lungs were fixed, photographed, preserved, and stained with H&E to analyze the presence of metastatic nodules. Immunohistochemistry (IHC) For immunohistochemistry, xenograft tumors from subcutaneous xenograft models were fixed with paraformaldehyde and paraffin-embedded. Slice the sample and install it on the slide. The sectioned slides were dewaxed in xylene, rehydrated with ethanol of decreasing concentration and subsequently microwave boiled in the antigen repair solution to expose the antigen, after that the slides were incubated by antibody against Ki-67, α-SMA or CD31 at 4°C overnight and corresponding horseradish peroxidase coupling with secondary antibodies for 10 min at room temperature. Next, diaminobenzidine was reacted under horseradish peroxidase catalyzation, brown pigments are formed at the site. Then, Nuclear staining with hematoxylin and examined under a microscope. The results of Ki-67 staining were analyzed by the positive staining cell. The IHC scores used to assess the results of Ki-67, α-SMA and CD31 staining, defined as percentage score (0 for 50%) × staining intensity score (none scored 0; weak scored 1; moderate scored 2; strong scored 3). The antibodies were listed in Supplementary Table 2. Statistical analysis GraphPad Prism software version 8.0 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA) were used for statistical analysis. All data of the study are presented as mean ± S.D. Statistical methods in this study included Student’s t test, one-way ANOVA, Chi-square test, Kaplan–Meier method, log-rank test and Pearson's correlation coefficient analysis and so on. Difference with P <0.05 was deemed to indicate statistically significant. Results LncRNA SZT2-AS1 is induced by hypoxia in a HIF-1-dependent manner in HCC To identify hypoxia-associated lncRNA in HCC, we initially performed RNA sequencing in Hep3B cells that were exposed to 20% O 2 or 1% O 2 for 24 hours. Then the differentially expressed lncRNA were obtained from the gene pool and the top 5 ones (SZT2-AS1, RP11-157L3.3, LOC643837, CTC-366B18.2, RP11-423O2.2) that were up-regulated in hypoxia condition based on fold-change were subjected to the further validation experiments (Fig. 1 A). These 5 lncRNA were validated in 6 kinds of HCC cell lines through RT-qPCR analysis, the data revealed that SZT2-AS1 was the only one which was consistently induced by hypoxia in all of the HCC cell lines (Fig. 1 B). Additionally, a time-dependent increase of SZT2-AS1 was observed both in Hep3B and MHCC97H cells that were exposed to hypoxia (Fig. 1 C, D). The stable HIF-1α knockdown subclones, HIF-2α knockdown subclones and DKD (both HIF-1α and HIF-2α knockdown) subclones were generated by using short hairpin RNA (shRNA)-expressing plasmids in Hep3B and MHCC97H cells. And the knockdown efficiencies were validated by Western blot (Fig. 1 E, F). Then, we found that the induction of SZT2-AS1 expression by hypoxia both in Hep3B and MHCC97H cells was abrogated by HIF-1α knockdown and DKD, while HIF-2α knockdown had no effect on SZT2-AS1 expression (Fig. 1 G, H). Furthermore, to determine whether HIF-1α directly bind to the SZT2-AS1 gene to activate its transcription, we applied ChIP assay in Hep3B and MHCC97H cells by using antibody against HIF-1α, HIF-2α or HIF-1β. The data indicated that hypoxia-induced binding of HIF-1 (HIF-1α + HIF-1β) to consensus HIF binding sites located 3.4 kb and 0.5 kb 5' to the transcription start site (TSS). The − 3.4kb site contained two consensus HIF binding site sequences as direct repeats separated by 51 base pairs (bp) (Fig. 1 I, J), whereas the − 0.5kb site contained a consensus HRE sequence (Fig. 1 K, L). Besides, the plasmids containing mutant HRE sites of SZT2-AS1 gene were established for the dual luciferase reporter assay and the data showed that HIF-1α overexpressing significantly increased the luciferase activity of wild type SZT2-AS1-HRE, rather than the mutant type (Fig. 1 M, N). In conclusion, our data suggests SZT2-AS1 is a HIF-1 target gene and hypoxia induces SZT2-AS1 expression in a HIF-1-dependent manner in HCC. SZT2-AS1 is an oncogene and prognostic biomarker for HCC Next, we attempted to explore the expression and clinical significance of SZT2-AS1 in HCC. Data analysis in TCGA from GEPIA platform ( http://gepia.cancer-pku.cn ) revealed that SZT2-AS1 was significantly increased in HCC (Fig. 2 A), which was consistent with the finding in HCC cell lines (Fig. 1 B) and our sample cohort (Fig. 2 B). Then, the patients in our cohort were divided into two subgroups based on the median expression of SZT2-AS1 in HCC tissues, and the survival curves were made with five years follow-up data. Kaplan–Meier analysis revealed that the patients in high SZT2-AS1 group obviously had a worse overall survival rate and disease-free survival rate compared to the low expression group (Fig. 2 C, D), and the similar results were also obtained from the GEPIA platform (Fig. 2 E, F). And the correlations between SZT2-AS1 and the clinicopathologic features were analyzed. Data showed that SZT2-AS1 expression was closely related to tumor size, venous infiltration and TNM stage (Table 1 ). In a word, the above findings demonstrated that SZT2-AS1 is an oncogene and potential prognostic biomarker for HCC. Table 1 Correlation between SZT2-AS1 expression and the clinicopathologic characteristics in HCC Characteristics Cases ( n = 125) Expression of SZT2-AS1 P High ( n = 63) Low ( n = 62) Age (year) ≤ 50 75 40 35 0.422 >50 50 23 27 Gender Male 96 52 44 0.125 Female 29 11 18 Serum AFP level (ng/mL) < 400 28 10 18 0.078 ≥ 400 97 53 44 Tumor size (cm) < 5 68 26 42 0.003** ≥ 5 57 37 20 Number of tumor nodules 1 107 53 54 0.636 ≥ 2 18 10 8 Venous infiltration Absent 11 9 2 0.029* Present 114 54 60 Edmondson-Steiner grading I + II 28 10 18 0.078 III + IV 97 53 44 TNM stage I + II 98 44 54 0.019* III + IV 27 19 8 HCC, hepatocellular carcinoma; HBV, hepatitis B virus;AFP, alpha-fetoprotein; TNM, tumor-node-metastasis.* P < 0.05,** P < 0.01 SZT2-AS1 promotes HCC cells growth, metastasis and angiogenesis in vitro The SZT2-AS1 knockdown subclones of Hep3B and MHCC97H were established and the efficiencies were confirmed by RT-qPCR (Fig. 3 A). Then, the cells growth ability was evaluated by MTT assay and EdU assay. The MTT assay data indicated that the viabilities were dramatically weakened in SZT2-AS1 knockdown subclones of Hep3B and MHCC97, compared the control groups (Fig. 3 B). Similarly, when SZT2-AS1 expression was repressed, the number of EdU positively stained cells was obviously reduced in both Hep3B and MHCC97H cells (Fig. 3 C). Meanwhile, the effects of SZT2-AS1 expression on migrated and invaded abilities were assed with Transwell migration and invasion assays as well as the wound-healing assay. As expected, in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H, much less cells passing through the chamber membrane were observed in Transwell migration and invasion assays (Fig. 3 D). In addition, wound healing abilities of Hep3B and MHCC97H were memorably decreased abrogated when the SZT2-AS1 was silenced (Fig. 3 E). And, the tube formation assay showed that the effect of promoting angiogenesis by SZT2-AS1was repressed by the SZT2-AS1 shRNAs (Fig. 3 F). Take together, we demonstrate that SZT2-AS1 promotes HCC cells growth, metastasis and angiogenesis in vitro. SZT2-AS1 promotes HCC cells growth, metastasis and angiogenesis in vivo To further validate the functions of SZT2-AS1 in HCC cells growth, metastasis and angiogenesis, we established the orthotopic HCC model, subcutaneous xenograft model and tail vein injection lung metastasis model in BALB/c nude mice with stable SZT2-AS1 knockdown subclones of Hep3B and MHCC97H cells. In the orthotopic HCC model, the H&E staining results in liver indicated that SZT2-AS1 knockdown suppressed the formation of tumor nodules in mouse liver (Fig. 4 A). In the subcutaneous xenograft model, the growth curves indicated that the tumor growth was significantly inhibited when SZT2-AS1 was knocked down, which was also proved by the final tumor weight comparation (Fig. 4 B). Furthermore, the immunohistochemistry staining of Ki-67 which represented the cells proliferation ability was conducted in the mouse tumor sections, and the weaker positive staining of CD31 and α-SMA was observed in all SZT2-AS1 knockdown groups compared to the respond control group (Fig. 4 C, D), demonstrating that SZT2-AS1 expression promotes breast cancer vascularization. Meanwhile, H&E staining was performed in the tail vein injection lung metastasis model to measure the tumor formation in lung. Obviously, the tumor formation in lung tissues was markedly repressed in SZT2-AS1 knockdown groups (Fig. 4 E). Thus, we conclude that SZT2-AS1 markedly promotes HCC cells growth, metastasis and angiogenesis in vivo. SZT2-AS1 mediates hypoxia-induced HCC progression As hypoxic condition boosts HCC progression, and SZT2-AS1 was found to be a hypoxia-induced gene here, then we attempted to assess whether SZT2-AS1 mediated the hypoxia-induced HCC progression. The rescue experiments in Hep3B and MHCC97H indicated that SZT2-AS1 was induced by hypoxia, whereas the induction was abrogated by SZT2-AS1 shRNA (Fig. 5 A). Then. MTT assay, EdU assay, tranwell assays, wound-healing assay and tube formation assay were conducted by using SZT2-AS1 knockdown subclones or the corresponding control subclones which were exposed to normoxia (20% O 2 ) or hypoxia (1% O 2 ). Hypoxia dramatically induced shNTC subclones viability and growth, whereas the cells growth could hardly be promoted by hypoxia with the presence of SZT2-AS1 knockdown, which were observed both in MTT assay (Fig. 5 B) and EdU assay (Fig. 5 C). Besides, in the Transwell assays (Fig. 5 D) and wound-healing assay (Fig. 5 E), the number of cells passing through chamber membrance and the wound healing progression were significantly increased by hypoxia, whereas the promotion effects were obviously reversed by SZT2-AS1 knockdown. we further explored whether SZT2-AS1 promoted HCC angiogenesis to facilitate HCC progression. The data of tube formation assay revealed that the tumor-conditioned medium (TCM) from hypoxia conditional HCC cells induced HUVECs to develop more and larger tubes than the TCM from normoxia conditional HCC cells, whereas the tube formation could hardly be increased by hypoxia with the presence of SZT2-AS1 knockdown (Fig. 5 F). Thus, we demonstrate that SZT2-AS1 mediates hypoxia-induced HCC progression. SZT2-AS1 mediates the HIF-1 heterodimer formation in HCC The GO biological process enrichment was further analyzed based on the above hypoxia-related RNA-seq data, and SZT2-AS1 was found to be in the gene cluster of GO item of Protein Heterodimerization activity, which was one of the top 10 GO items (Fig. 6 A). As it has been reported that some lncRNAs can act as the scaffold to mediate the binding interaction of proteins, and HIF-1 is a heterodimer consisting of HIF-1α and HIF-1β, so we proposed the hypothesis that SZT2-AS1 might function through acting as a scaffold to mediate the formation of HIF-1 heterodimer. Besides, we evaluated the subcellular localization of SZT2-AS1 in HCC cells. FISH assay and RNA subcellular location analysis showed that SZT2-AS1 was mainly located in the nuclear of Hep3B and MHCC97H cells no matter whether under hypoxia or not (Fig. 6 B, C), suggesting the nuclear-regulatory function of SZT2-AS1. Furthermore, RIP assay indicated that SZT2-AS1 was enriched by HIF-1α and HIF-1β under hypoxia (Fig. 6 D), meanwhile RNA pull-down assay indicated that HIF-1α and HIF-1β were enriched by SZT2-AS1 (Fig. 6 E), suggesting that SZT2-AS1 could bind to HIF-1α protein and HIF-1β protein. Unfortunately, SZT2-AS1 had no effect on neither the stability nor the nuclear translocation of HIF-1α, HIF-2αor HIF-1β in Hep3B and MHCC97H cells (Fig. 6 F, G). However, IP assay by using anti-HIF-1α with the presence of SZT2-AS1 shRNA indicated that the binding between HIF-1β and HIF-1α was blocked by SZT2-AS1 knockdown (Fig. 6 H). Besides, bimolecular fluorescence complementation assay based on complementation of split Renilla luciferase (Rluc) was applied to evaluate the interaction status between HIF-1α and HIF-1β. The results revealed that the binding between HIF-1α and HIF-1β was induced by hypoxia while abrogated with the presence of SZT2-AS1 shRNAs (Fig. 6 I). Subsequently, in order to delineate the structural determinants for the association between SZT2-AS1 and HIF-1α, RNA pull-down assays were performed with a series of SZT2-AS1 truncated fragments. The nucleotides 1–233nt and nucleotides 234-466nt regions were found to be associated with HIF-1α, meanwhile we also found the nucleotides 234-466nt and nucleotides 466-699nt regions were associated with HIF-1β (Fig. 6 J). Next, in order to determine the HIF-1α domain required for SZT2-AS1 binding, HIF-1α truncated fragments including bHLH, PAS-A, PAS-B and TAD domains were purified by GST-tag. RNA pull-down assay in vitro showed SZT2-AS1 strongly bound to the PAS-A and TAD domain of HIF-1α (Fig. 6 K). Collectively, the above findings demonstrate that SZT2-AS1 mediates the HIF-1 heterodimer formation in HCC. SZT2-AS1 acts as a coactivator for HIF-1 transcriptional activity in HCC under hypoxia Next, we attempted to detect whether SZT2-AS1 regulated HIF-1 transcriptional activity in HCC cells under hypoxia. RT-qPCR which were conducted in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H indicated that the HIF-1 targeting genes VEGFA, ANGPTL4 and PGF were induced by hypoxia, whereas the inductions were blocked by SZT2-AS1 knockdown, but not for the RPL13A gene (Fig. 7 A). We next determined whether SZT2-AS1 functioned through the effect on HIF-1 transactivation potency by constructing a HIF-1α reporter plasmid (HRE-FLuc) which containing three hypoxia-response elements (HREs) and firefly luciferase coding sequences was used for the promoter-activity assay. Date indicated that ectopic expression of HIF-1α or SZT2-AS1 alone enhanced HIF-1 transcriptional activity, whereas co-expression of HIF-1α and SZT2-AS1 synergistically enhanced the promoter activity (Fig. 7 B). In contrast, SZT2-AS1 knockdown resulted in a significant reduction in ectopic HIF-1a-induced HIF-1 transcriptional activation (Fig. 7 B). Furthermore, we next investigated whether the occupancy of HREs by HIFs was affected by SZT2-AS1 expression. ChIP assays revealed a significant decrease in the hypoxia-induced occupancy of the ANGPTL4, VEGFA and PGF HREs by HIF-1α and HIF-1β (but not HIF-2α) in SZT2-AS1-knockdown Hep3B (Fig. 7 C) and MHCC97H cells (Fig. 7 D). Thus, we conclude that SZT2-AS1 acts as a coactivator of HIF-1 for transcriptional activity in HCC under hypoxia. SZT2-AS1 is required for hypoxia-induced histone trimethylation at HREs As shown in Fig. 6 A, SZT2-AS1 was also found to be in the gene cluster of GO item of positive regulation of histone methylation, and histone trimethylation is one of the most common histone modifications at HREs, so we attempted to test whether SZT2-AS1 knockdown has any impact on histone trimethylation at HREs. Western blot indicated that hypoxia significantly increased the trimethylation levels of lysine 4 and 36 of H3 in NTC subclones but not in SZT2-AS1-knockdown subclones (Fig. 8 A). Next, we performed ChIP assays to analyze the trimethylation levels of H3K4 specifically at the ANGPTL4, VEGFA, and PGF HREs. The hypoxic induction of the trimethylation levels of H3K4 at the ANGPTL4, VEGFA, and PGF HREs were observed in NTC cells and were abrogated in SZT2-AS1-knockdown cells (Fig. 8 B, C). Similar results were observed for H3K36 (Fig. 8 D, E). In addition, the hypoxic inductions of H3K4me3 and H3K36me3 marks at the ANGPTL4, VEGFA, and PGF HREs were observed in NTC cells and were abrogated in HIF-1α-knockdown cells (Fig. 8 B-E). Collectively, the above findings reveal that HIF-mediated induction of SZT2-AS1 expression and recruitment of SZT2-AS1 to HREs leads to the hypoxia-induced trimethylation of histones H3 at HREs and increased HIF occupancy of HREs. SZT2-AS1 regulates H3K4me3 and H3K36me3 by recruiting SMYD2 As we have found that hypoxia-induced SZT2-AS1 regulated H3K4me3 and H3K36me3 in HCC cells, and given that histone methylation is usually mediated by histone methyltransferase, we sought to figure out whether SZT2-AS1 could directly bind to histone methyltransferase. Then, RNA pull-down and mass spectrometry were performed in Hep3B cells to identify the potential SZT2-AS1-associated proteins. SMYD2 was identified as a candidate binding protein which is a histone methyltransferase (Fig. 9 A). In addition, RNA pull-down assay was performed both in Hep3B and MHCC97H cells by using biotinylated SZT2-AS1 and antisense SZT2-AS1 to verify the finding. And the data consistently showed that SMYD2 bound to SZT2-AS1(Fig. 9 B). Besides, the RIP assays showed SZT2-AS1 was pulled down by anti-SMYD2 antibody (Fig. 9 C). And, neither SZT2-AS1 nor hypoxia had any effect on SMYD2 expression (Fig. 9 D). Furthermore, rescue assays were conducted to investigate whether SMYD2 mediated SZT2-AS1-regulated histone methylation. Western blot data showed that SZT2-AS1-knockdown decreased the levels of H3K4me3 and H3K36me3, and the reductions were reversed by overexpressing SMYD2 (Fig. 9 E). Subsequently, RNA pull-down assays were performed with a series of SZT2-AS1 truncated fragments to identify the structural determinants for the association between SZT2-AS1 and SMYD2. The nucleotides 234-466nt and nucleotides 467-699nt regions were found to be associated with SMYD2(Fig. 9 F). In conclusion, these findings suggest that SZT2-AS1 regulates H3K4me3 and H3K36me3 by recruiting SMYD2. Discussion The hypoxic microenvironment provides a favorable intrinsic environment for the solid cancer development and progression[ 25 ]. Although only a few hypoxia-related cancer targeted drugs have been applied in clinical practice currently, its broad development and application space can’t be questioned[ 26 – 29 ]. Once being exposed to hypoxia, cancer cells immediately initiate HIF system to modulate the target genes transcription, through which at least hundreds of genes are transcribed[ 29 , 30 ]. In virtue of the boom in transcriptome sequencing technique, a growing number of non-coding RNAs have been identified as HIF target genes[ 31 , 32 ]. Though lncRNA has no ability to encode protein, it possesses a variety of different mechanisms, that has been attracting researchers worldwide to explore the mystery box. And the vital functions of lncRNA in hypoxia-mediated cancer progression have been recognized recently and plenty of hypoxia-responsive lncRNAs have been identified[ 32 ]. For example, HIF-1α-activated KDM4A-AS1 promotes HCC progression via the miR-411-5p/ KPNA2/AKT pathway[ 23 ]. Hypoxia-induced MAPKAPK5-AS1 contributes to the growth and metastasis through the MAPKAPK5-AS1 and HIF-1α signaling loop[ 21 ]. And lncRNA PVT1 modulates nasopharyngeal carcinoma cell proliferation by stabilizing HIF-1α and activating KAT2A acetyltransferase[ 33 ]. Here, SZT2-AS1 was identified a potential hypoxia-induced gene by RNA sequencing in HCC cells. Then, we attempted to explore whether SZT2-AS1 was directly induced by HIFs, and ChIP assay and dual luciferase reporter gene assay make it clear that SZT2-AS1 is an only directly transcribed by HIF-1, rather than HIF-2 or both HIF-1 and HIF-2, suggesting that SZT2-AS1 is a HIF-1 target gene though some genes have been proved the both HIF-1 and HIF-2 co-targeting genes. Based on the previous studies, almost all of the hypoxia-responsive genes could be regulated by HIF-1, whereas some genes can only be regulated by HIF-2, and some can be regulated by both HIF-1 and HIF-2. Unfortunately, it seems that rare studies have described the difference of these regulation mechanisms clearly so far and further studies are required. Besides, in order to determine the expression of SZT2-AS1 in HCC tissues and cell lines. Interestingly, SZT2-AS1 is not only upregulated in HCC tissues, where the hypoxic microenvironment inherently exists especially in the region close to the central area of the tumor, but also in the HCC cell lines which were not exposed to the hypoxic condition. The findings make us speculate that there might have some other mechanisms that are associated with SZT2-AS1 overexpression in HCC and need to be further studied, but hypoxic response is at least one. For all of the cancers including HCC, early detection by identifying reliable and sensitive biomarkers is the essential step for timely treatment before reaching to advanced stage. And the advantages such as sensitivity, specificity, stability of lncRNA as biomarker for cancer diagnosis and prognosis have fostered the hope that a breakthrough may be near. In the present study, SZT2-AS1 is highly expressed in HCC and the expression of SZT2-AS1 is closely related to HCC growth, venous infiltration, TNM stage and prognosis. Furthermore, SZT2-AS1-knockdown inhibits HCC growth and metastasis, which was validated by cytological experiments and animal experiments. Thus, we demonstrate that SZT2-AS1 may be a promising HCC biomarker. However, expanding the sample size and multicenter study are required for further confirm the findings. Mechanistically, we identified a novel positive feedback loop mechanism that hypoxia-induced SZT2-AS1, in turn, through acting as a co-activator promotes the formation of HIF-1 heterodimer consisting HIF-1α and HIF-1β, the histone trimethylation, the occupancy of HIF-1 to HRE sites of HIF target genes, and the transcription of HIF-1 target genes, so SZT2-AS1 is a key component in the positive feedback loop of HIF-1 transactivation. More and more co-activators of HIFs have been reported recently, and the mechanisms are diverse[ 12 , 13 , 34 ]. By RNA sequencing, one novel HIF-1α co-activator ZMIZ1 is identified, which is also co-regulated by hypoxia and METTL4[ 35 ]. JMJD1A can interact with HIF-2α to form a co-activator complex, which binds to the HRE of EPO gene and increases EPO expression by catalyzing demethylation of H3K9me2[ 36 ]. TET1 is shown to be a transcriptional co-activator that interacts with HIF-1α and HIF-2α to enhance their transactivation activity independent of its enzymatic activity[ 37 ]. Thus, as to lncRNAs, the role of working as scaffolds to recruit proteins and mediate proteins binding confer them more possibility to act as the co-activators. Here, SZT2-AS1 is mainly located in nucleus which is identified as a scaffold to bring HIF-1α and HIF-1β together to form the HIF-1 heterodimer to further enhance gene transcription. Meanwhile, SZT2-AS1 promotes the histone trimethylation at HREs, resulting in chromatin decondensation to give much more space for occupancy of HIF-1 to HREs, while the HIF target genes transcriptions are accelerated. Directly binding relation between SZT2-AS1 and SMYD2 which is a H3K4 and H3K36 histone methyltransferase was verified by mass spectrometry (MS) and RNA pull down assay. SZT2-AS1 facilitated the methyltransferase activity of SMYD2 by directly binding and recruit it to the histidine residues at HREs, thus promoting the global patterns of H3K4me3 and H3K36me3 in HCC cells. We are the first to demonstrate that lncRNAs can directly influence the enzyme activity of H3 methyltransferase as effector molecules like coenzymes. Hence, both HIF-1α and SZT2-AS1 expression are correlated with HCC and patient prognosis. Therapies for patients with advanced HCC are not currently satisfactory. The present study demonstrates that pharmacologic inhibition of HIF-1 and/or SZT2-AS1 activity may provide novel therapeutic strategies for these patients. Declarations Ethics approval and consent to participate The present study was approved by the Ethics Committees of the First Affiliated Hospital of Xi’an Jiaotong University. Consent for publication Informed consent was obtained from all of the patients. Availability of data and materials The RNA-seq data generated by this study are publicly available in Gene Expression Omnibus (GEO) at GSE271612. Conflict of interest The authors declare no potential conflicts of interest. Funding This study was supported by grants from the National Natural Science Foundation of China (82103565)， the Natural Science basic Research Program of Shaanxi Province (2022JQ-756), the Fundamental Research Funds for the Central Universities (xzy012022093), and the Key Research and Development Program of Shaanxi, China (No.2024SF-YBXM-142). Authors' contributions RL, YG, QL and YW conceived and designed research. RL, YG, QL, GY, HT, YZ and WY performed research and collected data. J.-W.S., RL, YG, QL, GY, HT, YZ, WY, QL and YW analyzed data. RL YW wrote the manuscript with the help from other co-authors. References Bray, F., et al., Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Additional Declarations There is no duality of interest Supplementary Files S3.RawdataforWesternblot.pptx Table.S1.Primers.docx Table.S2.Primaryantibodyinformation.docx Cite Share Download PDF Status: Published Journal Publication published 22 Nov, 2024 Read the published version in Cell Death & Differentiation → Version 1 posted Editorial decision: revise 12 Sep, 2024 Review # 2 received at journal 06 Sep, 2024 Review # 1 received at journal 04 Aug, 2024 Reviewer # 2 agreed at journal 30 Jul, 2024 Reviewer # 1 agreed at journal 28 Jul, 2024 Reviewers invited by journal 27 Jul, 2024 Submission checks completed at journal 26 Jul, 2024 Editor assigned by journal 26 Jul, 2024 First submitted to journal 26 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4805397","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":332418344,"identity":"80f471cc-1e34-4d8f-a5fe-2d0ef0a9cd20","order_by":0,"name":"Yufeng Wang","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-7470-4938","institution":"the First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Yufeng","middleName":"","lastName":"Wang","suffix":""},{"id":332418345,"identity":"14f7881e-a25b-4a1f-b6ae-f2e268f8b53d","order_by":1,"name":"Runkun Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Runkun","middleName":"","lastName":"Liu","suffix":""},{"id":332418346,"identity":"be4e1d9f-eb80-4dcf-b0a2-a14540725941","order_by":2,"name":"Yixian Guo","email":"","orcid":"","institution":"the First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yixian","middleName":"","lastName":"Guo","suffix":""},{"id":332418347,"identity":"03a7f392-db2f-4077-a536-4ffde961848b","order_by":3,"name":"Guozhi Yin","email":"","orcid":"","institution":"the First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Guozhi","middleName":"","lastName":"Yin","suffix":""},{"id":332418348,"identity":"7f8199a9-4d4e-4b80-982f-ca8eca8f7987","order_by":4,"name":"Hang Tuo","email":"","orcid":"","institution":"the First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Tuo","suffix":""},{"id":332418349,"identity":"5a6a6551-fc1d-4c4f-959a-adade888d10d","order_by":5,"name":"Yifeng Zhu","email":"","orcid":"","institution":"the First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yifeng","middleName":"","lastName":"Zhu","suffix":""},{"id":332418350,"identity":"3a0306a2-cc28-4494-a5bf-0a376bdde70e","order_by":6,"name":"Wei Yang","email":"","orcid":"","institution":"the First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Yang","suffix":""},{"id":332418351,"identity":"9bf118fa-253a-45b6-847f-f2af6eebf951","order_by":7,"name":"Qingguang Liu","email":"","orcid":"","institution":"the First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Qingguang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-07-26 05:05:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4805397/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4805397/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41418-024-01419-x","type":"published","date":"2024-11-22T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63086363,"identity":"9868e916-3e6b-4ee9-a437-06a2dbc6430b","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":850161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLncRNA SZT2-AS1 is induced by hypoxia in a HIF-1-dependent manner in HCC.\u003c/strong\u003e (A)\u0026nbsp; Heat maps were prepared showing lncRNA levels in Hep3B cells exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 24h for hypoxia-induced and hypoxia-repressed lncRNAs. The top 5 hypoxia-induced lncRNAs ranked by fold-change were listed. (B) Fold changes of the top 5 lncRNAs in 6 different HCC cell lines exposed to hypoxia for 24h were shown. (C, D) Hep3B or MHCC97H cells were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 6h, 12h, 24h, 48h, and RT-qPCR was used to test SZT2-AS1 expression at different time point.***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus 0h (two-way ANOVA).(E, F) Hep3B and MHCC97H subclones expressing a nontargeting control (NTC) shRNA, an shRNA targeting HIF-1 or HIF-2, or shRNAs targeting both HIF-1 and HIF-2 [double knockdown (DKD)] were constructed. And the subclones were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 6 hours for Western blot. (G, H) Hep3B and MHCC97H subclones were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 24h followed by RT-qPCR. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus NTC at 20%,\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus NTC at 1% (two-way ANOVA). (I-L) Hep3B or MHCC97H subclones were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 16h and ChIP assays were performed. Primers encompassing HRE sites in SZT2-AS1 gene were used for qPCR. Results were normalized to IgG at 20% O\u003csub\u003e2\u003c/sub\u003e. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus group at 20% O\u003csub\u003e2 \u003c/sub\u003e(two-way ANOVA). (M, N) Plasmids containing wild type (WT) or mutant type (MUT) of HRE sites of SZT2-AS1 gene were established for the dual luciferase reporter assay.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/431e946db86d989a73aeaaa7.png"},{"id":63086361,"identity":"0198042d-6a2c-4e59-9487-5d6e82ebdf7f","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":448419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZT2-AS1 is an oncogene and prognostic biomarker for HCC. \u003c/strong\u003e(A) TCGA data from GEPIA platform (http://gepia.cancer-pku.cn) was used to analyze the expression of SZT2-AS1 in HCC. (B) SZT2-AS1 expression was tested in the cohort from our hospital. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (Student’s t test). (C) SZT2-AS1 expression was tested in human normal hepatocyte MIHA and HCC cell lines. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus MIHA\u003csub\u003e \u003c/sub\u003e(two-way ANOVA). (D, E) Kaplan–Meier analysis was applied to analyze SZT2-AS1 expression on 5-year overall survival rate and disease-free survival rate. (F, G) Data from GEPIA platform was used to analyze SZT2-AS1 expression on 5-year overall survival rate and disease-free survival rate.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/f65a0dde175b4065a05634bc.png"},{"id":63086805,"identity":"6c701523-0e6a-4b74-bb2a-8a2450631b83","added_by":"auto","created_at":"2024-08-23 03:15:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5840950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZT2-AS1 promotes HCC cells growth, metastasis and angiogenesis in vitro. \u003c/strong\u003e(A)\u0026nbsp; SZT2-AS1 knockdown subclones of Hep3B and MHCC97H were established and the efficiencies were confirmed by RT-qPCR. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus shNTC\u003csub\u003e \u003c/sub\u003e(two-way ANOVA). (B) MTT assay was conducted in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 versus shNTC (two-way ANOVA with Sidak’s t test). (C) EdU assay was conducted in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus shNTC\u003csub\u003e \u003c/sub\u003e(two-way ANOVA). (D) Transwell migration and invasion assay was conducted in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus shNTC\u003csub\u003e \u003c/sub\u003e(two-way ANOVA). (E) Wound healing assay was conducted in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus shNTC\u003csub\u003e \u003c/sub\u003e(two-way ANOVA). (F) Tube formation assay was conducted in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H. Branch number and total tube length were analyzed by Image J. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus shNTC\u003csub\u003e \u003c/sub\u003e(two-way ANOVA).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/c1c72396bdcf4526b5ca865e.png"},{"id":63086370,"identity":"1792dd1b-7673-479c-8890-9fd3c95c86f3","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12067047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZT2-AS1 promotes HCC cells growth, metastasis and angiogenesis in vivo. \u003c/strong\u003e(A) Orthotopic HCC model was constucted, and H\u0026amp;E staining was performed to test the formation of tumor nodules in mousr liver. (B) Subcutaneous xenograft model was constructed, then tumor volume was tested every 3 days to establish the growth curves, and the final tumor weight was measured. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus shNTC (two-way ANOVA with Sidak’s t test, two-way ANOVA). (C, D) Tumor sections from subcutaneous xenograft model were analyzed by immunohistochemistry for Ki-67, α-SMA and CD31. The stained area was quantified by ImageJ software. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus NTC (two-way ANOVA). （E）H\u0026amp;E staining was performed in the tail vein injection lung metastasis model to measure the tumor formation in lung. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus NTC (two-way ANOVA).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/0060231dcaf2ac6877e0fdd8.png"},{"id":63086366,"identity":"ec23c5fd-3874-459d-b56c-66fe8ffcde6e","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6601488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZT2-AS1 mediates hypoxia-induced HCC progression. \u003c/strong\u003e(A) RT-qPCR was used to test SZT2-AS1 expression in the indicated groups. Rescue experiments of MTT assay (B), EdU assay (C), Transwell assay (D), wound healing assay (E) and tube formation assay (F) were conduceted. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus hypoxia+shNTC (two-way ANOVA).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/98ca5498e002a05c308ab293.png"},{"id":63086368,"identity":"b37d9779-435b-4f21-bb85-7761159786db","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2043353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZT2-AS1 mediates the HIF-1 heterodimer formation in HCC. \u003c/strong\u003e(A) The GO biological process enrichment of hypoxia-related lncRNAs was analyzed based on the hypoxia-related RNA-seq data. The top 10 categories for each analysis were shown. RNA subcellular fraction assay (B) and FISH assay (C) were applied to determine the subcellular location of SZT2-AS1 in Hep3B and MHCC97H cells exposed to 20% O\u003csub\u003e2\u003c/sub\u003e or 1% O\u003csub\u003e2 \u003c/sub\u003efor\u003csub\u003e \u003c/sub\u003e24h. (D)RIP assay was conducted with the antibodies against IgG, HIF-1α，HIF-2α and HIF-1β. Then qPCR was performed to test SZT2-AS1 expression. \u0026nbsp;***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (Student’s t test). (E) Immunoblot detection of HIF-1α or HIF-1β retrieved by biotinylated SZT2-AS1 RNA pull-down assay. Sense or antisense SZT2-AS1 RNAs were incubated with 16 h hypoxia-treated Hep3B or MHCC97H nuclear extracts and then pulled down by streptavidin beads, followed by western blot analysis. (F) Western blot was used to test the effect of SZT2-AS1 knockdown on HIF-1α，HIF-2α and HIF-1β expression in Hep3B and MHCC97H cells exposed to 20% O\u003csub\u003e2\u003c/sub\u003e or 1% O\u003csub\u003e2 \u003c/sub\u003efor\u003csub\u003e \u003c/sub\u003e8h. (G) Immunoblot detection of HIF-1α, HIF-2α or HIF-1β in the nuclear extracts from the indicated Hep3B or MHCC97H subclones exposed to 20% O\u003csub\u003e2\u003c/sub\u003e or 1% O\u003csub\u003e2 \u003c/sub\u003efor\u003csub\u003e \u003c/sub\u003e24h. (H) SZT2-AS1 knockdown subclones and control were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 8h, and WCLs were prepared for immunoprecipitation using antibody against HIF-1α. (I) Bimolecular fluorescence complementation assay based on complementation of split Renilla luciferase (Rluc) was applied to evaluate the interaction status between HIF-1α and HIF-1β. (J) RNA pull-down assays were performed with a series of SZT2-AS1 truncated fragments to delineate the structural determinants for the association between SZT2-AS1 and HIF-1α. (K) SZT2-AS1 binding domain within HIF-1α. Schematic representation of HIF-1α functional domains and GST-HIF-1α variants are shown at the top. The Coomassie Blue staining showed loading of the proteins and arrowheads mark the GST-HIF-1α truncates. SZT2-AS1 were pulled down by GST-fusion proteins pre-bound on glutathione-Sepharose beads, followed by RT–qPCR detection of SZT2-AS1 retrieved as presented as percentage relative to input RNA. bHLH, basic helix–loop–helix; PAS, Per-ARNT-Sim; TAD, transactivation domain.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/793beb2219bf7b0300808f1d.png"},{"id":63086365,"identity":"3a622384-81c2-45b1-b650-2996a5d6f246","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":581031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZT2-AS1 acts as a coactivator for HIF-1 transcriptional activity in HCC under hypoxia. \u003c/strong\u003e(A) Hep3B and MHCC97H subclones were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 24 hours followed by RT-qPCR for HIF target genes VEGFA, ANGPTL4 and PGF. Non-HIF target gene RPL13A was analyzed as a negative control. Results were normalized to NTC at 20% O\u003csub\u003e2\u003c/sub\u003e. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus NTC at 20% O\u003csub\u003e2\u003c/sub\u003e; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus NTC at 1% O\u003csub\u003e2\u003c/sub\u003e (two-way ANOVA). (B) A plasmid containing HIF-1α-responsive luciferase reporter was co-transfected with empty vector, HIF-1α or pcDNA/SZT2-AS1 or shSZT2-AS1#1 into 293T cells for the reporter assay. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (two-way ANOVA). (C, D) Hep3B and MHCC97H subclones were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 16h, and ChIP assays were performed using antibody against IgG, HIF-1α, HIF-1βor HIF-2α and qPCR of VEGFA, ANGPTL4 and PGF HREs. Results were normalized to IgG at 20%O\u003csub\u003e2\u003c/sub\u003e shNTC\u003csub\u003e. \u003c/sub\u003e***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus NTC at 20% O\u003csub\u003e2\u003c/sub\u003e; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus NTC at 1% O\u003csub\u003e2\u003c/sub\u003e (two-way ANOVA).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/05c1926ba2b1e5a7212456f4.png"},{"id":63086369,"identity":"25e18960-319a-4b07-a62f-5cd050ec9709","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":793737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZT2-AS1 is required for hypoxia-induced histone trimethylation at HREs.\u003c/strong\u003e (A) Hep3B or MHCC97H subclones were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 48h, and Western blot were performed using antibody against H3K36me3 or H3K4me3. (B-E) Hep3B or MHCC97H subclones were exposed to 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 16h, and ChIP assays were performed using antibody against IgG, H3K36me3 or H3K4me3. Results were normalized to the first lane. \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 versus first lane; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus second lane (two-way ANOVA).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/99fc3014dcda79359d9fdd15.png"},{"id":63086806,"identity":"ea475645-80e5-45d4-817d-feff38212f4f","added_by":"auto","created_at":"2024-08-23 03:15:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":759755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZT2-AS1 regulates H3K4me3 and H3K36me3 by recruiting SMYD2.\u003c/strong\u003e (A) Identification of proteins associated with SZT2-AS1. The Coomassie Blue staining of RNA pull-down proteins was performed, and SZT2-AS1 enriched bands were analyzed by MS. And SMYD2 was identified. (B) Immunoblot detection of SMYD2retrieved by biotinylated SZT2-AS1 RNA pull-down assay. Sense or antisense SZT2-AS1 RNAs were incubated with 16 h hypoxia-treated Hep3B or MHCC97H nuclear extracts and then pulled down by streptavidin beads, followed by western blot analysis. （C）RIP assay was performed with anti-SMYD2 in Hep3B or MHCC97H, then RT-qPCR was applied to test SZT2-AS1 expression. ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 (Student’s t test). (D) Western blot was used to test the effect of SZT2-AS1 knockdown on SMYD2 expression. (E) Rescue experiments of Western blot was performed to test the effect of SZT2-AS1 knockdown on H3K4me3 and H3K36me3 levels. (F) RNA pull-down assays were performed with a series of SZT2-AS1 truncated fragments to delineate the structural determinants for the association between SZT2-AS1 and SMYD2.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/5c75b692beb13569994cb67b.png"},{"id":69609754,"identity":"9f2db724-2c7a-4fef-931b-239870d8e279","added_by":"auto","created_at":"2024-11-22 08:06:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35170359,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/745de70a-0fc8-4af3-8873-aa19fe14c387.pdf"},{"id":63086371,"identity":"150b81f4-7a82-4aa7-9f2c-4b5abdac1427","added_by":"auto","created_at":"2024-08-23 03:07:32","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":119262850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"S3.RawdataforWesternblot.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/29b7c7bb3a30cac3d4991847.pptx"},{"id":63086360,"identity":"1967c56c-26c0-4d96-aa6a-9c96c01cb637","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13830,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S1.Primers.docx","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/945586cf25f81696edd77de1.docx"},{"id":63086364,"identity":"75ff8be6-590d-4595-8e72-bae523506d77","added_by":"auto","created_at":"2024-08-23 03:07:31","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13108,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S2.Primaryantibodyinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4805397/v1/cdc41368d208ee29e2cbb401.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Hypoxia-induced SZT2-AS1 is required for HIF-1 heterodimer formation and histone trimethylation in HCC cells under hypoxic microenvironment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) holds the third position for cancer-related mortality within the globe and the second deadliest cancer in China[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The characteristics of high malignancy, high mortality and high recurrence rate of hepatocellular carcinoma (HCC) make the current treatment still very challenging. Though surgical resection remains the most effective and curative strategy, the fact is that most HCC patients have already missed the best operation stage for surgical resection when they are diagnosed, while gene target therapy would be recommended as one of the treatments to the advanced HCC[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, HCC progression is a complicated, multistep biological process, implicating multiple genes and biomolecules, which is a critical characteristic of all of the cancers. Therefore, in clinical practice, therapeutic effects of the current targeted drugs for HCC are still not very favorable[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. And uncovering the molecular mechanisms of HCC will facilitate the improvement of early HCC diagnosis precision and the development of more efficient therapeutic targets.\u003c/p\u003e \u003cp\u003eHypoxic microenvironment is an intrinsic trait of solid tumors, and is strongly associated with an invasive phenotype and poor prognosis in many cancer types[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The transcription factors hypoxia-inducible factors (HIFs) are a kind of pivotal regulator of response to hypoxic stress, among of which HIF-1 is the most common one and in charge of at least hundreds of protein coding and non-coding RNA genes, which are involved in multiple aspects of tumourigenesis, including proliferation, angiogenesis, invasion, metastasis, glucose and energy metabolism, and cancer stem-like properties[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. HIF is a heterodimer of an oxygen-regulated α subunit and a stably expressed β subunit, and the heterodimer formation is the cornerstone action for the occupancy of HIF to hypoxia response element (HRE) containing the core HIF binding sequence 5\u0026prime;-RCGTG-3\u0026prime; (R\u0026thinsp;=\u0026thinsp;A or G) in target genes and the initiation of its transcription process[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, a large body of evidence suggest that during the transcription process the co-activators of HIF are essential for HIF-mediated gene transcription, through binding to HIF-1α or HIF-1β or both to modulate the HIF protein stability, nuclear translocation, the occupancy of HIF to HRE, HIF activity or histone modifications[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It's worth noting that these co-activators not only include the proteins, such as FACT[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], NARF[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], NANOG[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], PADI4[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], some long non-coding RNAs like LncHIFCAR[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], DLEU1[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] are also identified as HIF-1 co-activators in recent years.\u003c/p\u003e \u003cp\u003eThough lncRNAs are a cluster RNAs that have no ability to encode protein and are more than 200nt length, their diverse functional mechanism that are totally different from coding-genes make them the hot pint[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The mechanism of lncRNAs involved in included regulated gene transcription (in cis or trans), chromatin remodel, and mRNA splicing; produced endogenous small interfering RNAs (siRNAs) and microRNA precursors; change protein localization; regulate protein activity; and organized protein components[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].In our previous researches, plenty of lncRNAs associated with HCC progression have been identified. For instance, lncRNA CASC2 inhibits epithelial-mesenchymal transition (EMT) in HCC cells through the CASC2/miR-367/FBXW7 axis[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, our study investigated that MCM3AP-AS1 promotes HCC growth by targeting the miR-194-5p/FOXA1 axis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recently, especially with the booming development of high-throughput technology, more and more lncRNAs have been indicated to be related to tumor hypoxic microenvironment. For example, MAPKAPK5-AS1 expression is upregulated under hypoxia and promoted HCC progression via miR-154-5p/PLAGL2/HIF-1α signaling loop[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. RUNX1-IT1 is restrained by hypoxia-induce histone deacetylase 3 to inhibits cancer stem-like properties and HCC cells proliferation[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. KDM4A-AS1, which is induced by oxygen deficiency, promotes the growth of HCC and transition through the KDM4A-AS1/ KPNA2/HIF-1α signal circuit[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, much more mechanism about hypoxia-related lncRNA remain further work to figure out.\u003c/p\u003e \u003cp\u003eIn this study, hypoxia-conditional RNA-seq in HCC cells was performed to scan and identify a cluster of hypoxia-regulated lncRNAs, where the novel hypoxia-induced lncRNA SZT2-AS1 was focused. Then, SZT2-AS1 was demonstrated to be induced by hypoxia in a HIF-1-dependent manner. In addition, the expression of SZT2-AS1 was upregulated in HCC and closely associated with clinical prognosis in HCC. Functionally, SZT2-AS1 promoted HCC growth, metastasis and angiogenesis, and mediated the hypoxia-induced HCC progression. Furthermore, in turn, SZT2-AS1 recruited HIF-1α and HIF-1β to form the HIF-1 heterodimer, meanwhile promoting the trimethylation modification of histones (H3K4me3 and H3K36me3) at HRE sites via recruiting SMYD2 to make chromatin decondensation, then increasing the occupancy of HIF-1 to HRE and HIF-1 transcriptional activity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue samples\u003c/h2\u003e \u003cp\u003eTotal 125 pairs of HCC tissue samples and adjacent non-tumor tissue samples, which were histopathologically confirmed, were collected from HCC patients stood hepatectomy in the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University. Neither chemotherapy nor radiotherapy was administered before surgery to any of the patients. A temperature of -80\u0026deg;C was used to store all samples. Our study got approval from the Ethics Committees of the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University, all patients provided informed consent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe human normal liver cell line (MIHA), six cell lines (HepG2, Huh7, Hep3B, MHCC97H and SK-Hep-1) and human embryonic kidney (HEK) 293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All of the cells were maintained in incubator (37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e), and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Invitrogen, CA, USA). For hypoxia treatment, physical hypoxic condition (1%O\u003csub\u003e2\u003c/sub\u003e) was generated by Forma Series II 3130 incubator (Thermo Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids and cell transfection\u003c/h2\u003e \u003cp\u003eThe small hairpin RNAs (shRNAs) targeting SZT2-AS1 (shSZT2-AS1#1, shSZT2-AS1#2) were obtained from GeneCreate Biological Engineering Co., Ltd. (Wuhan, China). HIF-1α, HIF-2α shRNAs and scrambled shRNA (shNTC) were purchased from GeneCopoeia (Guangzhou, China). The human HIF-1α or SMYD2 ORF cDNA clone (ov-HIF-1α, ov-SMYD2), and control empty vector were purchased from GeneCopoeia, Inc. All sequences were verified by DNA Sanger sequencing. Lentiviral production was achieved by transfecting target plasmids with psPAX2 packaging plasmid and pMD2.G envelope plasmids into HEK293T cells. Viral supernatant added to HCC cells with 8 \u0026micro;g/ml polybrene (Beyotime Biotech Inc., Shanghai, China), The transfected cells were treated with 3 \u0026micro;g/ml puromycin and 100 \u0026micro;g/ml Ampicillin to select knockdown and overexpressed single clone cells. Cell transfections were performed by using Lipofectamine 3000 reagent (Invitrogen, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eBy using Trizol (Thermo Fisher Scientific) after completing the designated intervention, the RNA from HCC cells and tissues was extracted. A reverse transcription kit was used to reverse-transcribe total RNA into cDNA (Invitrogen, CA, USA). Real-time qPCR analysis was performed using SYBR Green Premix PCR Master Mix (Roche Diagnostics, Mannheim, Germany). Normalizing the relative expression level to 18S and the expression was calculated by 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e methods. The sequences of the primers used are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot and immunoprecipitation (IP) assays\u003c/h2\u003e \u003cp\u003eRIPA buffer was used to isolate total protein from cells which supplemented with proteinase inhibitors and phosphatase inhibitors (Beyotime, Hangzhou, China). According to the manufacturer's instructions, BCA Protein assay kits (ZHHC Biotech Inc., Shaanxi, China) were used to determine protein concentration. Protein was separated by 10% or 8% or 15% concentration SDS-PAGE gels, then transferred to 0.22\u0026micro;m PVDF membranes (Millipore, Billerica, MA, USA). Following 3 hours of blocking by 10% nonfat milk, primary antibodies were used to incubate membranes at 4℃ temperature overnight. Afterwards, secondary antibodies conjugated to HRP were incubated on the membranes at room temperature for 1 hour. The blots were detected using enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA). For immunoprecipitation, equal amounts of WCLs (500 \u0026micro;g) were incubated with primary antibody HIF-1α (2 \u0026micro;g) in the presence of protein G-Sepharose beads (Amersham Biosciences) at 4\u0026deg;C overnight, and the immunoprecipitates were subjected to SDS-PAGE and immunoblot assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTranswell migration and invasion assays\u003c/h2\u003e \u003cp\u003eAs we previously reported[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], after completing the specified experimental processing, cell migration and invasion ability were investigated using Transwell migration and invasion assays following established protocols from prior research.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eWound healing assay was used to detect cell migration ability. After transfected HCC cells with different plasmid or virus, the experiment was conducted according to the protocols as we previously reported[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay and EdU assay\u003c/h2\u003e \u003cp\u003eLike MTT assay, specified transfected cells were added into 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/ well. Then with 0, 24, 48, and 72h after seeding, 20\u0026micro;L MTT solution (Sigma, USA) was added to each well and continued incubate for 4h at 37℃. After removed the supernatants, added 100 \u0026micro;l DMSO to each well. Each absorbance was measured at 490 nm by a microplate reader (Bio-Rad, Richmond, CA). For EdU assay, Cell-Light\u0026trade; EdU Apollo\u0026reg;567 (RiboBio Co., Ltd. Guangzhou, China) was used to evaluate of cell proliferation. Briefly, transfected HCC cells were cultured in 96-well plates. Then complete the experiment according to the protocol supply by manufacturer. The percentage of EdU positive cells was calculated using ImageJ software. EdU positive rate is calculated by counting at least five random fields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTube formation assay\u003c/h2\u003e \u003cp\u003eThe different treatment HCC cell culture medium was changed to serum-free DMEM medium for 48 h and then was collected, centrifuged and filtered to obtain tumor-conditioned medium (TCM). Prepare the dissolved Matrigel and precool the 24-well plate and pipets at -20℃. The precool 24-well plate was laid with 200\u0026micro;l Matrigel matrix (Corning Inc., Corning, NY, USA) incubated 37℃ for 30min. HUVEC (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e) cells were added to each well with 200\u0026micro;l TCM which came from HCC cells and supplemented with 10% FPS, and then incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 8h. Pictures were taken under a bright-field microscope and the capillary tubes were quantified by counting branch number and total tube length with Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe SZT2-AS1 promoter region sequence containing wild-type (WT) or mutated (MUT) sequences of hypoxia response elements (HREs) were embeded into pGL3-based vectors (Promega, USA). Luciferase reporter plasmid pGL3-based vectors expressing SZT2-AS1-WT or MUT were co-transfected into Hep3B and MHCC97H cells in 96-well plates with empty vector or pcDNA3.1/HIF-1α. After 24h, cells lysates were collected, the dual-luciferase reporter system kit (Beyotime, Shanghai, China) were used to measure Renilla and firefly luciferase activities on a microplate reader according to the manufacturer\u0026rsquo;s advices and protocols. Normalized luciferase activity according to The Renilla luciferase internal control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBimolecular fluorescence complementation assay\u003c/h2\u003e \u003cp\u003eThe pCMV-NRluc and pCMV-CRluc plasmids were constructed. The human bHLH-PAS domain of HIF-1α (amino acid residues 12\u0026ndash;396) was prepared by PCR amplification and the PCR product was cloned downstream of the N-terminal segment (residues 1-229) of Rluc, in pCMV-NRluc-HIF-1α\u003csub\u003e12\u0026minus;396\u003c/sub\u003e. Similarly, the bHLH-PAS domain of HIF-1β (residues 11\u0026ndash;510) was amplified and inserted upstream of the C-terminal segment (residues 230\u0026ndash;311) of Rluc, in pCMV-HIF-1β\u003csub\u003e11\u0026minus;510\u003c/sub\u003e-CRluc. MHCC97H and Hep3B cells were seeded at 2x10\u003csup\u003e5\u003c/sup\u003e cells per well of a 24-well plate and incubated for 24h. Cells were co-transfected with 300 ng of NRluc-HIF-1α\u003csub\u003e12\u0026minus;396\u003c/sub\u003e, 300 ng of HIF-1β\u003csub\u003e11\u0026minus;510\u003c/sub\u003e-CRluc, and 80 ng of pGL2-promoter, using Fugene-6 (Roche) according to the manufacturer\u0026rsquo;s instructions. Following 7h incubation, cells were treated with indicated shNTC or shZST2-AS1 for 24 h. Cells were then lysed and analyzed for the ratio of Rluc/Fluc using the Dual Luciferase Assay System (Promega).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eHep3B cells were seeded into six-well plates for 24h, and then incubated in 20%O\u003csub\u003e2\u003c/sub\u003e or 1%O\u003csub\u003e2\u003c/sub\u003e for another 24h. Total RNA was isolated from the cells using TRIzol (Invitrogen) and treated with deoxyribonuclease (Qiagen). Library preparation and sequencing using the NovaSeq 5000 platform (Illumina) were performed. The FASTQ files were subjected to quality check and analyzed by Genialis Inc. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genialis.com\u003c/span\u003e\u003cspan address=\"https://www.genialis.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Differential expression results with a false discovery rate of \u0026lt;\u0026thinsp;0.05 and mRNA fold change of \u0026gt;\u0026thinsp;1.5 were used as acut off for further downstream analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation (RIP)\u003c/h2\u003e \u003cp\u003eRIP assay was conducted by utilizing the EZ-Magna-RIP kit (MilliporeSigma, Birlington, MA) according to the the manufacturer's instructions. Cells were lysed by lysis buffer which added with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA) and RNase inhibitors (Millipore Sigma). Then, the cell lysate was pre-washed with recombinant protein A/G agarose (Thermo Fisher Scientific) to reduce non-specific binding for 30 min at 4\u0026deg;C. One to twenty percent of the cell lysate were used as input. Then the specific antibody (anti-HIF-1α, anti-HIF-1β, anti-HIF-2α or IgG) and protein A/G magnetic beads were placed in equal amount of cell lysates for overnight at 4℃, negative control is used IgG. At the next day, RNA was eluted from the precipitated complex by using Trizol and transcribed into cDNA. RT-qPCR assay was performed to detect binding of RNA (SZT2-AS1) to proteins or antibody. The primary antibodies were listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNA pull-down assay\u003c/h2\u003e \u003cp\u003eThe interaction between SZT2-AS1 and SMYD2 or HIF-1α or HIF-1β was predicted with RNA-Protein Interaction Prediction (RPISeq). Biotin labelled Sense and antisense of SZT2-AS1 RNA were in vitro transcribed with AmpliScribe T7-Flash Biotin-RNA Transcription Kit (Epicentre), treated with RNase-free DNase I and purified with a RNeasy Mini Kit (Qiagen). To establish the appropriate secondary structure, biotinylated SZT2-AS1 RNA supplied with RNA structure buffer (10mM Tris pH7, 0.1M KCl and 10 mM MgCl2) was first heated up to 90\u0026deg;C for 2 min, then incubated on ice for 2 min and last transferred to room temperature (RT) for 20 min. The RNA was then mixed with hypoxic Hep3B and MHCC97H cells extract or purified proteins and incubated at RT for 1 h, followed by incubating with Streptavidin Mag Sepharose (GE Healthcare) at RT for 1 h. After follow-up wash, extract the pull-down complexes were analysed by standard western blot technique.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA Subcellular fraction\u003c/h2\u003e \u003cp\u003e To determine the cellular localization of SZT2-AS1, cytoplasmic and nuclear fractions were isolated and collected with the PARIS Kit (Life Technologies, Inc., Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Thereafter, collections from HCC cells both cytoplasm and nucleus were extracted out total RNA and cDNA was synthesized for the evaluation of SZT2-AS1. Briefly, we collected 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells and washed in PBS three times, and then 300\u0026micro;l Cell Fractionation Buffer resuspended cells and incubated at 4\u0026deg;C for 10 min. After 12000rpm centrifugation, aspiration of supernatant containing cytoplasmic components and 300\u0026micro;l Cell Disruption Buffer were resuspended collection of centrifugal precipitation containing nuclear fragments. The manufacturer\u0026rsquo;s instructions were used to extract RNA from the buffer containing cytoplasmic/nuclear fraction, following RT-PCR analysis of the levels of nuclear control transcript (U6), cytoplasmic control transcript (GAPDH) and SZT2-AS1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProtein isolation and analysis\u003c/h2\u003e \u003cp\u003eNuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) was using to protein isolation according to the manufacturer\u0026rsquo;s instructions. Briefly, cells that have been treated in advance were harvested and dissociated in 200\u0026micro;l Reagent mixture containing 1 mM PMSF (Key Gen BioTech, Nanjing, China). The dissociated cell was incubated at 4\u0026deg;C for 15 min. Then, adding 10\u0026micro;l Reagent B and vortex the mixture for 5 s with 1 min ice bath, after that centrifugation at 16000 g, for 5 min. The supernatant we collected was cytoplasmic protein and 50\u0026micro;l nuclear protein extraction reagent containing 1 mM PMSF were further resuspend the precipitation. After being vortexed and 4\u0026deg;C in turn for 30 min, the mixture was centrifugated 5 min at 16000 g for 4\u0026deg;C, and the supernatant was stored as nuclear protein. The subcellular fractions were determined by using BCA protein assay kit, and then subjected to immunoblotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePurification of GST-HIF-1α and In vitro RNA-binding assay\u003c/h2\u003e \u003cp\u003eLuria\u0026ndash;Bertani medium with ampicillin (50\u0026micro;g/ml) used to culture Escherichia coli host BL21(DE3) harbouring the expression vector pGEX-6p-1-HIF-1a, and induced by 0.3 mM IPTG at 30\u0026deg;C for 16 h. Affinity purification of Recombinant protein by Pierce Glutathione Super Flow Agarose (Pierce) following the manufacturer\u0026rsquo;s instructions. SZT2-AS1 RNA was synthesized in vitro and added to RNA structural buffer (10 mM Tris pH 7, 0.1 M KCl and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e), mixture first heated to 90\u0026deg;C for 2 min, follow incubated on ice for 2 min and then transfer to RT for 20 min to form the proper secondary RNA structure. GST fusion proteins on 20\u0026micro;l glutathione Sepharose beads were incubated with 2 mg SZT2-AS1 RNA synthesized in vitro which included in 50\u0026micro;l of RNA-binding buffer (0.1% NP-40, 100 mM KCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol and ribonuclease inhibitor) for 30 min at 4\u0026deg;C. Follow up, the glutathione Sepharose beads were washed with RNA-binding buffer three times to remove non-attached RNAs. Trizol reagent were used to extract the RNA samples retained on the beads and detected expression by RT\u0026ndash;qPCR. Calculate the relative retention value of the input RNA level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eMHCC97H and Hep3B cells were incubated at 20 or 1% O\u003csub\u003e2\u003c/sub\u003e for 16 hours and harvested for ChIP assay, cells cross-linked in 3.7% formaldehyde for 15 min, neutralized in 0.125M glycine for 5 min, and then used SDS lysis buffer to lysed HCC cells. Chromatin was sheared by sonication to an average length of 200\u0026ndash;1000 bp, and salmon sperm DNA/protein A agarose slurry (Millipore) precleared cells lysates for 1 hour, protein A\u0026ndash;agarose beads incubated with antibody against HIF-1α, HIF-2α, SMYD2, HIF-1β, H3K4me3, H3K36me3, H3 overnight at 4\u0026deg;C. Follow by serial washing of the agarose beads with low-salt, high-salt, and LiCl buffers, 1% SDS with 0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e elute DNA from beads, and reverse cross-links by addition of 0.2M NaCl. Purification of DNA by phenol-chloroform extraction and ethanol precipitation and amplified by RT-qPCR using primers listed in Supplementary Table\u0026nbsp;1. And antibodies were listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRNA fluorescent in situ hybridization (FISH)\u003c/h2\u003e \u003cp\u003eFISH kit (RiboBio, Guangzhou, China) was used to detected the Subcellular localization of SZT2-AS1 according to the manufacturer\u0026rsquo;s procedure. In brief, MHCC97H and Hep3B cells were cultured on glass coverslips into 24-well plates. After 4% paraformaldehyde was utilized to fix HCC cells and washed with PBS, then subjected to permeabilization (0.5%Triton-X100 PBS). HCC cells were incubated with prehybridization solution and hybridized with hybridization solution, and then incubated with hybridization solution contain Cy3-labeled SZT2-AS1 oligonucleotide probe overnight. HCC cells nuclei were visualized with DAPI. All images were captured and recorded under a Zeiss fluorescence photomicroscope (Carl Zeiss AG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eThe growth and metastasis ability of cells in vivo were assessed by the orthotopic HCC model, subcutaneous xenograft model and lung metastasis model in mice. 4 weeks old male BALB/C nude mice purchased from the Centre of Laboratory Animals at The Medical College of Xi\u0026rsquo;an Jiao tong University and the mice were randomly grouped (n\u0026thinsp;=\u0026thinsp;5 per group). All the animal experiments were approved by the Research Ethics Committee of Xi\u0026rsquo;an Jiao tong University.\u003c/p\u003e \u003cp\u003eFor the construction of orthotopic HCC model, 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e Hep3B-shSZT2-AS1 or MHCC97H-shSZT2-AS1 or control subclones were dissolved in 0.1 mL of DMEM culture medium. Mice were anesthetized with 3% pentobarbital sodium, and the liver was exposed by open surgery. The cells were injected into the liver of the nude mice, and the wound was sutured with 5\u0026thinsp;\u0026minus;\u0026thinsp;0 silk thread. 4 weeks later, the mice were sacrificed in accordance with ethical procedures to observe the tumor formation, and the liver tissues were used for H\u0026amp;E staining.\u003c/p\u003e \u003cp\u003eIn vivo subcutaneously tumor growth assay, 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e Hep3B-shSZT2-AS1 or MHCC97H-shSZT2-AS1 subclones and the same numbers corresponding control subclones were transplanted into the flank of 4-week-old BALB/c nude mice via subcutaneous injection. After injection, we measured tumor size calculated 0.5 \u0026times; length \u0026times; width \u0026times; width every 3 days. The nude mice were killed after 21 days in different groups, and the tumor specimens were weighed, fixed and harvested for IHC experiments.\u003c/p\u003e \u003cp\u003eIn vivo lung metastasis model, we intravenously injected 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells into the lateral tail vein of nude mice. After 5 weeks killed the mice and collect the lung tissues. Thereafter, the lungs were fixed, photographed, preserved, and stained with H\u0026amp;E to analyze the presence of metastatic nodules.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eFor immunohistochemistry, xenograft tumors from subcutaneous xenograft models were fixed with paraformaldehyde and paraffin-embedded. Slice the sample and install it on the slide. The sectioned slides were dewaxed in xylene, rehydrated with ethanol of decreasing concentration and subsequently microwave boiled in the antigen repair solution to expose the antigen, after that the slides were incubated by antibody against Ki-67, α-SMA or CD31 at 4\u0026deg;C overnight and corresponding horseradish peroxidase coupling with secondary antibodies for 10 min at room temperature. Next, diaminobenzidine was reacted under horseradish peroxidase catalyzation, brown pigments are formed at the site. Then, Nuclear staining with hematoxylin and examined under a microscope. The results of Ki-67 staining were analyzed by the positive staining cell. The IHC scores used to assess the results of Ki-67, α-SMA and CD31 staining, defined as percentage score (0 for \u0026lt;\u0026thinsp;5%; 1 for 5\u0026ndash;25%; 2 for 25\u0026ndash;50%; 3 for \u0026gt;\u0026thinsp;50%) \u0026times; staining intensity score (none scored 0; weak scored 1; moderate scored 2; strong scored 3). The antibodies were listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism software version 8.0 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA) were used for statistical analysis. All data of the study are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. Statistical methods in this study included Student\u0026rsquo;s t test, one-way ANOVA, Chi-square test, Kaplan\u0026ndash;Meier method, log-rank test and Pearson's correlation coefficient analysis and so on. Difference with \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was deemed to indicate statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eLncRNA SZT2-AS1 is induced by hypoxia in a HIF-1-dependent manner in HCC\u003c/h2\u003e \u003cp\u003eTo identify hypoxia-associated lncRNA in HCC, we initially performed RNA sequencing in Hep3B cells that were exposed to 20% O\u003csub\u003e2\u003c/sub\u003e or 1% O\u003csub\u003e2\u003c/sub\u003e for 24 hours. Then the differentially expressed lncRNA were obtained from the gene pool and the top 5 ones (SZT2-AS1, RP11-157L3.3, LOC643837, CTC-366B18.2, RP11-423O2.2) that were up-regulated in hypoxia condition based on fold-change were subjected to the further validation experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These 5 lncRNA were validated in 6 kinds of HCC cell lines through RT-qPCR analysis, the data revealed that SZT2-AS1 was the only one which was consistently induced by hypoxia in all of the HCC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, a time-dependent increase of SZT2-AS1 was observed both in Hep3B and MHCC97H cells that were exposed to hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). The stable HIF-1α knockdown subclones, HIF-2α knockdown subclones and DKD (both HIF-1α and HIF-2α knockdown) subclones were generated by using short hairpin RNA (shRNA)-expressing plasmids in Hep3B and MHCC97H cells. And the knockdown efficiencies were validated by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F). Then, we found that the induction of SZT2-AS1 expression by hypoxia both in Hep3B and MHCC97H cells was abrogated by HIF-1α knockdown and DKD, while HIF-2α knockdown had no effect on SZT2-AS1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H). Furthermore, to determine whether HIF-1α directly bind to the SZT2-AS1 gene to activate its transcription, we applied ChIP assay in Hep3B and MHCC97H cells by using antibody against HIF-1α, HIF-2α or HIF-1β. The data indicated that hypoxia-induced binding of HIF-1 (HIF-1α\u0026thinsp;+\u0026thinsp;HIF-1β) to consensus HIF binding sites located 3.4 kb and 0.5 kb 5' to the transcription start site (TSS). The \u0026minus;\u0026thinsp;3.4kb site contained two consensus HIF binding site sequences as direct repeats separated by 51 base pairs (bp) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J), whereas the \u0026minus;\u0026thinsp;0.5kb site contained a consensus HRE sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, L). Besides, the plasmids containing mutant HRE sites of SZT2-AS1 gene were established for the dual luciferase reporter assay and the data showed that HIF-1α overexpressing significantly increased the luciferase activity of wild type SZT2-AS1-HRE, rather than the mutant type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM, N). In conclusion, our data suggests SZT2-AS1 is a HIF-1 target gene and hypoxia induces SZT2-AS1 expression in a HIF-1-dependent manner in HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSZT2-AS1 is an oncogene and prognostic biomarker for HCC\u003c/h2\u003e \u003cp\u003eNext, we attempted to explore the expression and clinical significance of SZT2-AS1 in HCC. Data analysis in TCGA from GEPIA platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) revealed that SZT2-AS1 was significantly increased in HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which was consistent with the finding in HCC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and our sample cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Then, the patients in our cohort were divided into two subgroups based on the median expression of SZT2-AS1 in HCC tissues, and the survival curves were made with five years follow-up data. Kaplan\u0026ndash;Meier analysis revealed that the patients in high SZT2-AS1 group obviously had a worse overall survival rate and disease-free survival rate compared to the low expression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D), and the similar results were also obtained from the GEPIA platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). And the correlations between SZT2-AS1 and the clinicopathologic features were analyzed. Data showed that SZT2-AS1 expression was closely related to tumor size, venous infiltration and TNM stage (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In a word, the above findings demonstrated that SZT2-AS1 is an oncogene and potential prognostic biomarker for HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between SZT2-AS1 expression and the clinicopathologic characteristics in HCC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCases\u003c/p\u003e \u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;125)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eExpression of SZT2-AS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;62)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAge (year)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.422\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSerum AFP level\u003c/p\u003e \u003cp\u003e(ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTumor size (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.003**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNumber of\u003c/p\u003e \u003cp\u003etumor nodules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.636\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVenous infiltration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.029*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEdmondson-Steiner\u003c/p\u003e \u003cp\u003egrading\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u0026thinsp;+\u0026thinsp;II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u0026thinsp;+\u0026thinsp;IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTNM stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u0026thinsp;+\u0026thinsp;II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.019*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u0026thinsp;+\u0026thinsp;IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eHCC, hepatocellular carcinoma; HBV, hepatitis B virus;AFP, alpha-fetoprotein; TNM, tumor-node-metastasis.*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05,**\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eSZT2-AS1 promotes HCC cells growth, metastasis and angiogenesis in vitro\u003c/h2\u003e \u003cp\u003eThe SZT2-AS1 knockdown subclones of Hep3B and MHCC97H were established and the efficiencies were confirmed by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Then, the cells growth ability was evaluated by MTT assay and EdU assay. The MTT assay data indicated that the viabilities were dramatically weakened in SZT2-AS1 knockdown subclones of Hep3B and MHCC97, compared the control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similarly, when SZT2-AS1 expression was repressed, the number of EdU positively stained cells was obviously reduced in both Hep3B and MHCC97H cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Meanwhile, the effects of SZT2-AS1 expression on migrated and invaded abilities were assed with Transwell migration and invasion assays as well as the wound-healing assay. As expected, in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H, much less cells passing through the chamber membrane were observed in Transwell migration and invasion assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In addition, wound healing abilities of Hep3B and MHCC97H were memorably decreased abrogated when the SZT2-AS1 was silenced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). And, the tube formation assay showed that the effect of promoting angiogenesis by SZT2-AS1was repressed by the SZT2-AS1 shRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Take together, we demonstrate that SZT2-AS1 promotes HCC cells growth, metastasis and angiogenesis in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eSZT2-AS1 promotes HCC cells growth, metastasis and angiogenesis in vivo\u003c/h2\u003e \u003cp\u003eTo further validate the functions of SZT2-AS1 in HCC cells growth, metastasis and angiogenesis, we established the orthotopic HCC model, subcutaneous xenograft model and tail vein injection lung metastasis model in BALB/c nude mice with stable SZT2-AS1 knockdown subclones of Hep3B and MHCC97H cells. In the orthotopic HCC model, the H\u0026amp;E staining results in liver indicated that SZT2-AS1 knockdown suppressed the formation of tumor nodules in mouse liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In the subcutaneous xenograft model, the growth curves indicated that the tumor growth was significantly inhibited when SZT2-AS1 was knocked down, which was also proved by the final tumor weight comparation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Furthermore, the immunohistochemistry staining of Ki-67 which represented the cells proliferation ability was conducted in the mouse tumor sections, and the weaker positive staining of CD31 and α-SMA was observed in all SZT2-AS1 knockdown groups compared to the respond control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D), demonstrating that SZT2-AS1 expression promotes breast cancer vascularization. Meanwhile, H\u0026amp;E staining was performed in the tail vein injection lung metastasis model to measure the tumor formation in lung. Obviously, the tumor formation in lung tissues was markedly repressed in SZT2-AS1 knockdown groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Thus, we conclude that SZT2-AS1 markedly promotes HCC cells growth, metastasis and angiogenesis in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSZT2-AS1 mediates hypoxia-induced HCC progression\u003c/h3\u003e\n\u003cp\u003eAs hypoxic condition boosts HCC progression, and SZT2-AS1 was found to be a hypoxia-induced gene here, then we attempted to assess whether SZT2-AS1 mediated the hypoxia-induced HCC progression. The rescue experiments in Hep3B and MHCC97H indicated that SZT2-AS1 was induced by hypoxia, whereas the induction was abrogated by SZT2-AS1 shRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Then. MTT assay, EdU assay, tranwell assays, wound-healing assay and tube formation assay were conducted by using SZT2-AS1 knockdown subclones or the corresponding control subclones which were exposed to normoxia (20% O\u003csub\u003e2\u003c/sub\u003e) or hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e). Hypoxia dramatically induced shNTC subclones viability and growth, whereas the cells growth could hardly be promoted by hypoxia with the presence of SZT2-AS1 knockdown, which were observed both in MTT assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and EdU assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Besides, in the Transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and wound-healing assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), the number of cells passing through chamber membrance and the wound healing progression were significantly increased by hypoxia, whereas the promotion effects were obviously reversed by SZT2-AS1 knockdown. we further explored whether SZT2-AS1 promoted HCC angiogenesis to facilitate HCC progression. The data of tube formation assay revealed that the tumor-conditioned medium (TCM) from hypoxia conditional HCC cells induced HUVECs to develop more and larger tubes than the TCM from normoxia conditional HCC cells, whereas the tube formation could hardly be increased by hypoxia with the presence of SZT2-AS1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Thus, we demonstrate that SZT2-AS1 mediates hypoxia-induced HCC progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eSZT2-AS1 mediates the HIF-1 heterodimer formation in HCC\u003c/h2\u003e \u003cp\u003eThe GO biological process enrichment was further analyzed based on the above hypoxia-related RNA-seq data, and SZT2-AS1 was found to be in the gene cluster of GO item of Protein Heterodimerization activity, which was one of the top 10 GO items (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As it has been reported that some lncRNAs can act as the scaffold to mediate the binding interaction of proteins, and HIF-1 is a heterodimer consisting of HIF-1α and HIF-1β, so we proposed the hypothesis that SZT2-AS1 might function through acting as a scaffold to mediate the formation of HIF-1 heterodimer. Besides, we evaluated the subcellular localization of SZT2-AS1 in HCC cells. FISH assay and RNA subcellular location analysis showed that SZT2-AS1 was mainly located in the nuclear of Hep3B and MHCC97H cells no matter whether under hypoxia or not (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C), suggesting the nuclear-regulatory function of SZT2-AS1. Furthermore, RIP assay indicated that SZT2-AS1 was enriched by HIF-1α and HIF-1β under hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), meanwhile RNA pull-down assay indicated that HIF-1α and HIF-1β were enriched by SZT2-AS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), suggesting that SZT2-AS1 could bind to HIF-1α protein and HIF-1β protein. Unfortunately, SZT2-AS1 had no effect on neither the stability nor the nuclear translocation of HIF-1α, HIF-2αor HIF-1β in Hep3B and MHCC97H cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G). However, IP assay by using anti-HIF-1α with the presence of SZT2-AS1 shRNA indicated that the binding between HIF-1β and HIF-1α was blocked by SZT2-AS1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Besides, bimolecular fluorescence complementation assay based on complementation of split Renilla luciferase (Rluc) was applied to evaluate the interaction status between HIF-1α and HIF-1β. The results revealed that the binding between HIF-1α and HIF-1β was induced by hypoxia while abrogated with the presence of SZT2-AS1 shRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Subsequently, in order to delineate the structural determinants for the association between SZT2-AS1 and HIF-1α, RNA pull-down assays were performed with a series of SZT2-AS1 truncated fragments. The nucleotides 1\u0026ndash;233nt and nucleotides 234-466nt regions were found to be associated with HIF-1α, meanwhile we also found the nucleotides 234-466nt and nucleotides 466-699nt regions were associated with HIF-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Next, in order to determine the HIF-1α domain required for SZT2-AS1 binding, HIF-1α truncated fragments including bHLH, PAS-A, PAS-B and TAD domains were purified by GST-tag. RNA pull-down assay in vitro showed SZT2-AS1 strongly bound to the PAS-A and TAD domain of HIF-1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Collectively, the above findings demonstrate that SZT2-AS1 mediates the HIF-1 heterodimer formation in HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eSZT2-AS1 acts as a coactivator for HIF-1 transcriptional activity in HCC under hypoxia\u003c/h2\u003e \u003cp\u003eNext, we attempted to detect whether SZT2-AS1 regulated HIF-1 transcriptional activity in HCC cells under hypoxia. RT-qPCR which were conducted in SZT2-AS1 knockdown subclones of Hep3B and MHCC97H indicated that the HIF-1 targeting genes VEGFA, ANGPTL4 and PGF were induced by hypoxia, whereas the inductions were blocked by SZT2-AS1 knockdown, but not for the RPL13A gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We next determined whether SZT2-AS1 functioned through the effect on HIF-1 transactivation potency by constructing a HIF-1α reporter plasmid (HRE-FLuc) which containing three hypoxia-response elements (HREs) and firefly luciferase coding sequences was used for the promoter-activity assay. Date indicated that ectopic expression of HIF-1α or SZT2-AS1 alone enhanced HIF-1 transcriptional activity, whereas co-expression of HIF-1α and SZT2-AS1 synergistically enhanced the promoter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In contrast, SZT2-AS1 knockdown resulted in a significant reduction in ectopic HIF-1a-induced HIF-1 transcriptional activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Furthermore, we next investigated whether the occupancy of HREs by HIFs was affected by SZT2-AS1 expression. ChIP assays revealed a significant decrease in the hypoxia-induced occupancy of the ANGPTL4, VEGFA and PGF HREs by HIF-1α and HIF-1β (but not HIF-2α) in SZT2-AS1-knockdown Hep3B (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) and MHCC97H cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Thus, we conclude that SZT2-AS1 acts as a coactivator of HIF-1 for transcriptional activity in HCC under hypoxia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eSZT2-AS1 is required for hypoxia-induced histone trimethylation at HREs\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, SZT2-AS1 was also found to be in the gene cluster of GO item of positive regulation of histone methylation, and histone trimethylation is one of the most common histone modifications at HREs, so we attempted to test whether SZT2-AS1 knockdown has any impact on histone trimethylation at HREs. Western blot indicated that hypoxia significantly increased the trimethylation levels of lysine 4 and 36 of H3 in NTC subclones but not in SZT2-AS1-knockdown subclones (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Next, we performed ChIP assays to analyze the trimethylation levels of H3K4 specifically at the ANGPTL4, VEGFA, and PGF HREs. The hypoxic induction of the trimethylation levels of H3K4 at the ANGPTL4, VEGFA, and PGF HREs were observed in NTC cells and were abrogated in SZT2-AS1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, C). Similar results were observed for H3K36 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, E). In addition, the hypoxic inductions of H3K4me3 and H3K36me3 marks at the ANGPTL4, VEGFA, and PGF HREs were observed in NTC cells and were abrogated in HIF-1α-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-E). Collectively, the above findings reveal that HIF-mediated induction of SZT2-AS1 expression and recruitment of SZT2-AS1 to HREs leads to the hypoxia-induced trimethylation of histones H3 at HREs and increased HIF occupancy of HREs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eSZT2-AS1 regulates H3K4me3 and H3K36me3 by recruiting SMYD2\u003c/h2\u003e \u003cp\u003eAs we have found that hypoxia-induced SZT2-AS1 regulated H3K4me3 and H3K36me3 in HCC cells, and given that histone methylation is usually mediated by histone methyltransferase, we sought to figure out whether SZT2-AS1 could directly bind to histone methyltransferase. Then, RNA pull-down and mass spectrometry were performed in Hep3B cells to identify the potential SZT2-AS1-associated proteins. SMYD2 was identified as a candidate binding protein which is a histone methyltransferase (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). In addition, RNA pull-down assay was performed both in Hep3B and MHCC97H cells by using biotinylated SZT2-AS1 and antisense SZT2-AS1 to verify the finding. And the data consistently showed that SMYD2 bound to SZT2-AS1(Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Besides, the RIP assays showed SZT2-AS1 was pulled down by anti-SMYD2 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). And, neither SZT2-AS1 nor hypoxia had any effect on SMYD2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). Furthermore, rescue assays were conducted to investigate whether SMYD2 mediated SZT2-AS1-regulated histone methylation. Western blot data showed that SZT2-AS1-knockdown decreased the levels of H3K4me3 and H3K36me3, and the reductions were reversed by overexpressing SMYD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). Subsequently, RNA pull-down assays were performed with a series of SZT2-AS1 truncated fragments to identify the structural determinants for the association between SZT2-AS1 and SMYD2. The nucleotides 234-466nt and nucleotides 467-699nt regions were found to be associated with SMYD2(Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). In conclusion, these findings suggest that SZT2-AS1 regulates H3K4me3 and H3K36me3 by recruiting SMYD2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe hypoxic microenvironment provides a favorable intrinsic environment for the solid cancer development and progression[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Although only a few hypoxia-related cancer targeted drugs have been applied in clinical practice currently, its broad development and application space can\u0026rsquo;t be questioned[\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Once being exposed to hypoxia, cancer cells immediately initiate HIF system to modulate the target genes transcription, through which at least hundreds of genes are transcribed[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In virtue of the boom in transcriptome sequencing technique, a growing number of non-coding RNAs have been identified as HIF target genes[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Though lncRNA has no ability to encode protein, it possesses a variety of different mechanisms, that has been attracting researchers worldwide to explore the mystery box. And the vital functions of lncRNA in hypoxia-mediated cancer progression have been recognized recently and plenty of hypoxia-responsive lncRNAs have been identified[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. For example, HIF-1α-activated KDM4A-AS1 promotes HCC progression via the miR-411-5p/ KPNA2/AKT pathway[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Hypoxia-induced MAPKAPK5-AS1 contributes to the growth and metastasis through the MAPKAPK5-AS1 and HIF-1α signaling loop[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. And lncRNA PVT1 modulates nasopharyngeal carcinoma cell proliferation by stabilizing HIF-1α and activating KAT2A acetyltransferase[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Here, SZT2-AS1 was identified a potential hypoxia-induced gene by RNA sequencing in HCC cells. Then, we attempted to explore whether SZT2-AS1 was directly induced by HIFs, and ChIP assay and dual luciferase reporter gene assay make it clear that SZT2-AS1 is an only directly transcribed by HIF-1, rather than HIF-2 or both HIF-1 and HIF-2, suggesting that SZT2-AS1 is a HIF-1 target gene though some genes have been proved the both HIF-1 and HIF-2 co-targeting genes. Based on the previous studies, almost all of the hypoxia-responsive genes could be regulated by HIF-1, whereas some genes can only be regulated by HIF-2, and some can be regulated by both HIF-1 and HIF-2. Unfortunately, it seems that rare studies have described the difference of these regulation mechanisms clearly so far and further studies are required.\u003c/p\u003e \u003cp\u003eBesides, in order to determine the expression of SZT2-AS1 in HCC tissues and cell lines. Interestingly, SZT2-AS1 is not only upregulated in HCC tissues, where the hypoxic microenvironment inherently exists especially in the region close to the central area of the tumor, but also in the HCC cell lines which were not exposed to the hypoxic condition. The findings make us speculate that there might have some other mechanisms that are associated with SZT2-AS1 overexpression in HCC and need to be further studied, but hypoxic response is at least one. For all of the cancers including HCC, early detection by identifying reliable and sensitive biomarkers is the essential step for timely treatment before reaching to advanced stage. And the advantages such as sensitivity, specificity, stability of lncRNA as biomarker for cancer diagnosis and prognosis have fostered the hope that a breakthrough may be near. In the present study, SZT2-AS1 is highly expressed in HCC and the expression of SZT2-AS1 is closely related to HCC growth, venous infiltration, TNM stage and prognosis. Furthermore, SZT2-AS1-knockdown inhibits HCC growth and metastasis, which was validated by cytological experiments and animal experiments. Thus, we demonstrate that SZT2-AS1 may be a promising HCC biomarker. However, expanding the sample size and multicenter study are required for further confirm the findings.\u003c/p\u003e \u003cp\u003eMechanistically, we identified a novel positive feedback loop mechanism that hypoxia-induced SZT2-AS1, in turn, through acting as a co-activator promotes the formation of HIF-1 heterodimer consisting HIF-1α and HIF-1β, the histone trimethylation, the occupancy of HIF-1 to HRE sites of HIF target genes, and the transcription of HIF-1 target genes, so SZT2-AS1 is a key component in the positive feedback loop of HIF-1 transactivation. More and more co-activators of HIFs have been reported recently, and the mechanisms are diverse[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. By RNA sequencing, one novel HIF-1α co-activator ZMIZ1 is identified, which is also co-regulated by hypoxia and METTL4[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. JMJD1A can interact with HIF-2α to form a co-activator complex, which binds to the HRE of EPO gene and increases EPO expression by catalyzing demethylation of H3K9me2[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. TET1 is shown to be a transcriptional co-activator that interacts with HIF-1α and HIF-2α to enhance their transactivation activity independent of its enzymatic activity[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, as to lncRNAs, the role of working as scaffolds to recruit proteins and mediate proteins binding confer them more possibility to act as the co-activators. Here, SZT2-AS1 is mainly located in nucleus which is identified as a scaffold to bring HIF-1α and HIF-1β together to form the HIF-1 heterodimer to further enhance gene transcription.\u003c/p\u003e \u003cp\u003eMeanwhile, SZT2-AS1 promotes the histone trimethylation at HREs, resulting in chromatin decondensation to give much more space for occupancy of HIF-1 to HREs, while the HIF target genes transcriptions are accelerated. Directly binding relation between SZT2-AS1 and SMYD2 which is a H3K4 and H3K36 histone methyltransferase was verified by mass spectrometry (MS) and RNA pull down assay. SZT2-AS1 facilitated the methyltransferase activity of SMYD2 by directly binding and recruit it to the histidine residues at HREs, thus promoting the global patterns of H3K4me3 and H3K36me3 in HCC cells. We are the first to demonstrate that lncRNAs can directly influence the enzyme activity of H3 methyltransferase as effector molecules like coenzymes.\u003c/p\u003e \u003cp\u003eHence, both HIF-1α and SZT2-AS1 expression are correlated with HCC and patient prognosis. Therapies for patients with advanced HCC are not currently satisfactory. The present study demonstrates that pharmacologic inhibition of HIF-1 and/or SZT2-AS1 activity may provide novel therapeutic strategies for these patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was approved by the Ethics Committees of the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all of the patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq data generated by this study are publicly available in Gene Expression Omnibus (GEO) at GSE271612.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (82103565)，\u0026nbsp;the Natural Science basic Research Program of Shaanxi Province (2022JQ-756), the Fundamental Research Funds for the Central Universities (xzy012022093), and the Key Research and Development Program of Shaanxi, China (No.2024SF-YBXM-142).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRL, YG, QL and YW conceived and designed research. RL, YG, QL, GY, HT, YZ and WY performed research and collected data. J.-W.S., RL, YG, QL, GY, HT, YZ, WY, QL and YW analyzed data. RL YW wrote the manuscript with the help from other co-authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray, F., et al., \u003cem\u003eGlobal cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.\u003c/em\u003e CA Cancer J Clin, 2024. \u003cstrong\u003e74\u003c/strong\u003e(3): p. 229-263.\u003c/li\u003e\n\u003cli\u003eWang, J., et al., \u003cem\u003eDiagnostic and Prognostic Value of Protein Post-translational Modifications in Hepatocellular Carcinoma.\u003c/em\u003e J Clin Transl Hepatol, 2023. \u003cstrong\u003e11\u003c/strong\u003e(5): p. 1192-1200.\u003c/li\u003e\n\u003cli\u003eZheng, Y., et al., \u003cem\u003eThe progress of immune checkpoint therapy in primary liver cancer.\u003c/em\u003e Biochim Biophys Acta Rev Cancer, 2021. \u003cstrong\u003e1876\u003c/strong\u003e(2): p. 188638.\u003c/li\u003e\n\u003cli\u003eVogel, A. and A. 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Hypoxia-inducible factors (HIFs), which are canonical transcription factors in response to hypoxia, are stabilized under hypoxia and they coordinate the process of hypoxia-induced gene expression leading to cancer progression. Increasing evidence has indicated that long noncoding RNAs (lncRNAs) which are closely associated with cancer play crucial roles in hypoxia-mediated HCC progression, while the mechanisms are largely unknown. Here, we identified a novel lncRNA SZT2-AS1 in HCC, which was induced by hypoxia in a HIF-1-dependent manner and promoted HCC growth, metastasis and angiogenesis. The clinical data indicated that SZT2-AS1 level was substantially upregulated in HCC and significantly associated with poor clinical outcomes, and acted as an independent prognostic predictor. Mechanistically, SZT2-AS1, in turn, recruited HIF-1α and HIF-1β to form the HIF-1 heterodimer. And SZT2-AS1 was required for the occupancy of HIF-1 to hypoxia response elements (HREs) and HIF target gene transcription. In addition, SZT2-AS1 was required for hypoxia-induced histone trimethylation (H3K4me3 and H3K36me3) at HREs. Through recruiting methyltransferase SMYD2, SZT2-AS1 promoted trimethylation modification of H3K4 and H3K36 in HCC cells. Taken together, our results uncovered a lncRNA-involved positive feedback mechanism under hypoxia and established the clinical value of SZT2-AS1 in prognosis and potential therapeutic strategy for HCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSignificance: \u003c/strong\u003eLncRNA SZT2-AS1 involves in a positive feedback mechanism under hypoxia, which provides a therapeutic strategy for HCC.\u003c/p\u003e","manuscriptTitle":"Hypoxia-induced SZT2-AS1 is required for HIF-1 heterodimer formation and histone trimethylation in HCC cells under hypoxic microenvironment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-23 03:07:26","doi":"10.21203/rs.3.rs-4805397/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-09-12T09:52:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-07T02:57:13+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-05T02:44:02+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-30T22:39:43+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-29T00:36:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-07-27T05:17:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-26T09:08:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-26T05:02:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2024-07-26T05:02:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"049b6718-4ebb-499a-a348-bfc9e55cd9de","owner":[],"postedDate":"August 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35200814,"name":"Biological sciences/Molecular biology/Epigenetics"},{"id":35200815,"name":"Biological sciences/Cancer/Cancer microenvironment"}],"tags":[],"updatedAt":"2024-11-22T08:05:47+00:00","versionOfRecord":{"articleIdentity":"rs-4805397","link":"https://doi.org/10.1038/s41418-024-01419-x","journal":{"identity":"cell-death-and-differentiation","isVorOnly":false,"title":"Cell Death \u0026 Differentiation"},"publishedOn":"2024-11-22 05:00:00","publishedOnDateReadable":"November 22nd, 2024"},"versionCreatedAt":"2024-08-23 03:07:26","video":"","vorDoi":"10.1038/s41418-024-01419-x","vorDoiUrl":"https://doi.org/10.1038/s41418-024-01419-x","workflowStages":[]},"version":"v1","identity":"rs-4805397","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4805397","identity":"rs-4805397","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}