Super-enhancer-associated LINC00963 promotes epithelial-mesenchymal transition in gastric cancer

Super-enhancer-associated LINC00963 promotes epithelial-mesenchymal transition in gastric cancer | 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 Research Article Super-enhancer-associated LINC00963 promotes epithelial-mesenchymal transition in gastric cancer Hong Du, Tingting Xiang, Ying Xia, Yong Jin, Fahua Deng, Wansong Xia, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4020742/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background In clinical practice, gastric cancer (GC) is a common malignancy with high morbidity. Accumulating research has revealed that lncRNAs are involved in the development and metastasis of tumor tissues in multiple cancers. As reported, LINC00963, a typical lncRNA is aberrantly expressed in gastric cancer. However, the underlying mechanisms of super-enhancers mediating remain unclear. Materials and methods The GC cell line enhancer-super-enhancer data were downloaded and analyzed from the NCBI database (GSE75595). Combined RT-qPCR and Sanger sequencing were employed to identify three variants of LINC00963 in gastric cell lines and peripheral blood samples from gastric cancer patients. The gene expression was detected through RT-qPCR, and their encoded protein expression levels were mesured by western blot. Transwell assayswere applied to assess the cell invasion and migration, while the apoptosis rate was tested by flow cytometry. A xenograft model was applied to simulate the tumor development process, during which the effect of LINC00963 on promoting tumorigenesis were investigated. Results Analysis of the GC cell line enhancer-super-enhancer data revealed a high expression of LINC00963 driven by super-enhancer. The variant 1 and variant 2 of LINC00963 exhibited high expression in GC cell line and the peripheral blood of gastric cancers. After down-regulated variant 1 of LINC00963, the results showed a significant increase in cell apoptosis rate. LINC00963 expression in GC cell line reduced upon exposure to a low dose of the bromodomain and extra-terminal inhibitor, JQ1, which resulted in a decrease of the protein levels of β-catenin and ZEB1. As a result, the protein expression levels of several marker proteins related to epithelial-mesenchymal transition (EMT), such as Vimentin, N-cadherin and Snail were observed to decrease, which may lead to an inhibition on GC cells metastasis, thereby suppressing tumor growth. Conclusion This study demonstrated that the Super-enhancer-associated LINC00963 is via a Wnt/β-catenin pathway that promotes EMT and tumor metastasis in gastric cancer. Gastric cancer Super enhancer LINC00963 Epithelial-mesenchymal transition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 4 Figure 5 1. Introduction In clinical practice, gastric cancer (GC) is a common malignancy with high morbidity. According to the Global Cancer Report, in 2020, GC ranks 5th in amount of new cancer cases (5.6% of 19.3 million), and 4th in cancer-related deaths (7.7% of 10 million) [ 1 ]. Invasion and metastasis are the main reasons for the low survival rate and poor prognosis of GC patients[ 2 , 3 ]. As a consequence, it is important to elucidate the molecular mechanisms of GC invasion and metastasis in order to identify effective GC prognostic molecular markers and corresponding therapeutic targets. A super-enhancer (SE) is a genomic region in mammals, often spanning an average size of up to 30 kb, that comprises multiple enhancers[ 4 ]. These enhancers work collectively to recruit core regulatory circuitry transcription factors (TFs), thus becoming an important cause of the mediated transcriptional dysregulation which is observed in human cancers [ 5 , 6 ]. Super-enhancers regulate the abnormal expression of specific genes to promote the malignant process of tumors[ 7 ] in pancreatic cancer[ 8 ], colorectal cancer[ 9 ], Ewing sarcoma [ 10 ], and GC[ 11 ]. It has also been reported that super-enhancers are more sensitive to drugs, such as JQ1 and THZ1, than typical enhancers [ 12 ]. Low doses of JQ1 can interfere with super-enhancer function, thereby inhibiting the abnormal expression of the related downstream target genes, most of which are specific genes that can identify cells. As a result, the occurrence and development of tumors were suppressed [ 7 , 13 , 14 ].. Enhancer-mediated genes have been reported to promote tissue invasion, such as myocyte enhancer factor 2C (MEF2C)[ 15 ] [ 1 ]. Our previous research has identified several lncRNAs associated with the occurrence, development, and metastasis of gastric cancer [ 16 , 17 ]. We speculate that since SE can modulate specific genes expression to promote the process of tumor development, it is also conceivable that SEs may similarly regulate the lncRNAs expression, resulting in the promotion of malignant progression. However, there have been few reports on super-enhancer-associated genes regulating invasion and migration in GC to date. This work investigated the function and its underlying mechanisms of a SE-associated gene LINC00963 in promoting GC progression with in vivo, in vitro experiments and clinical cases. The underlying molecular mechanisms were also elucidated that SE-associated LINC00963 promotes GC invasion and migration through the Wnt/β-catenin pathway. The results indicate that LINC00963 is a potential marker for labeling the metastasis of GC. 2. Materials and methods 2.1 Cells cultivation Gastric cancer cell lines (AGS, MKN45, HGC-27, and MGC803) and an immortalized gastric epithelial cell line (GES-1) were purchased from the National Infrastructure of Cell Line Resource of China (Beijing, China). All cell lines were cultured in RPMI-1640 medium (Gibco, Carlsbad, California, USA) containing 10% fetal bovine serum (Lonza Science SRL, Basel, Switzerland) and 1% penicillin-streptomycin (Invitrogen, Shanghai, China) and incubated under 5% CO 2 at 37°C. Short tandem repeats (STRs) of cell lines and Mycoplasma contamination were checked regularly. 2.2 human GC samples The peripheral blood of 30 patients with GC and the peripheral blood of 30 healthy individuals at the time of physical examinations were collected 2mL from GC patients and healthy individuals in the Affiliated Hospital of Guizhou Medical University (Guizhou, China). The blood samples were labeled and stored at − 80°C. The collection of peripheral blood samples was authorized and approved by the Ethics Committee of the Affiliated Hospital of Guizhou Medical University (2020 [106]). Blood samples are taken with the verbal consent of the patient and family. 2.3 Super-enhancer data analysis of GC cell lines The GC cell line enhancer-super-enhancer data is available through GEO under accession number GSE75595 (Accessed in 2018). The GC cell line enhancer-super-enhancer data is available through GEO under accession number GSE75595 (Accessed in 2019). Gene Ontology (GO) enrichment analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway database ( http://www.genome.jp/ ) to describe the super-enhancer target gene function. To predict enhancer-super-enhancer target gene sets, we performed biological pathway enrichment analysis based on the KEGG database, referencing the comprehensive data analysis of excellent enhancers in the previous work reported by Whyte WA and Wang J[ 4 ]. The Cancer Cell Line Encyclopedia (CCLE) database ( www.broadinstitute.org/ccle ) was used to comprehensively analyze the gene expression of GC cell lines, particularly the LINC00963 gene expression. 2.4 RNA extraction and RT- qPCR analysis Total RNA was extracted from peripheral blood and GC cell line using the UNlQ-10 column Trizol RNA extraction kit (B511321-0100; Sangon Biotech Co., Ltd., Shanghai, China). Expression of the target genes was quantified with RT-qPCR kits (Takara, Cat. DRR820A), normalized to the housekeeping gene HPRT. The relative gene expression levels were determined by the comparative threshold cycle (2 −ΔΔCT ) method. The primer sequences of RT-qPCR are listed in Supplementary Table 1. 2.5 Small interfering RNA (siRNA) transfection and lentivirus vector construction The full-length sequences of LINC00963 (NR_038955.1) and the newly discovered LINC00963-V1 were obtained according to the NCBI database. Specific siRNAs for LINC00963-V1 were designed using siRNA technology, and then synthesized by Shanghai Gene Pharma Co., Ltd. (Shanghai, China). The siRNA sequences are listed in Supplementary Table 2 . Lipofectamine® RNAi MAX reagent (Invitrogen, Shanghai, China) was used to transfect siRNA. The cell lines of stable LINC00963 knockdown were established by lentivirus-mediated delivery of LINC00963-specific shRNA (sh-LINC00963). Lentivirus packaging kit GM easy™ (Genomeditech, Shanghai, China) was used to synthesize lentivirus. Lentivirus vector is pPLK plasmid sh-LINC00963 (public protein/plasmid library). The shRNA sequences are listed in Supplement Table 3 . 2.6 Migration and invasion assays Transwell determination using 8.0 µm pore size Transwell permeable carrier (Corning, New York, USA) was used. The number of cells used for cell migration and invasion determination was 5 × 10 4 and 6 × 10 4 , respectively. After the experiment, they were fixed in methanol and stained with crystal violet solution for photography[ 18 ] . 2.7 Apoptosis assay Apoptotic cells were assayed by the cell apoptosis assay kit (Beyotime Biotechnology, Shanghai, China). Briefly, HGC-27 or MGC803 cells were seeded into 6-well plates and transfected with a negative control siRNA (si-NC) or LINC00963 siRNA. After a 72-h transfection, 5 × 10 5 cells were collected from each group and washed twice with cold PBS. Binding buffer (500 µl) was added to resuspend the cells in each sample tube. Annexin V-TITC (5 µl) was added and gently mixed, then incubated at room temperature for 15 min in the dark. PI (10 µl) was added and incubated on ice for 5 min in the dark and detected with flow cytometry. 2.8 Immunoblotting assay Immunoblotting assay was performed according to our previous studies[ 17 , 19 ]. The following antibodies are used: GAPDH (Bioworld, Nanjing, China; 1:5000); E-cadherin (ab40772, Abcam, Boston, Massachusetts, USA; 1:1000); N-cadherin (ab76011, Abcam; 1:1000); vimentin (ET1610-39, Huabio, Hangzhou, China; 1:20000); MMP7 (ab205525, Abcam; 1:1000); β-catenin (ab32572, Abcam; 1:400); Zeb1 (WL03489, Wanleibio, Shenyang, China; 1:2000); Snail (WL01863, Wanleibio; 1:2000); and horseradish peroxidase-labeled goat anti-rabbit IgG (Multi Sciences, Hangzhou, China; 1:5000). 2.9 In vivo experiments Female BALB/c nude mice (Age, 5weeks; Weight, 20 ± 3g) were purchased from the SLRC Laboratory Animal Center (Shanghai, China). A dose 1×106 HGC-27 cells from sh-LINC00963 were subcutaneously injected into the right inguinal region and sh-NC were injected into the left inguinal region of BALB/c nude mice. A total of 5 mice. The tumor volume and body weight were monitored every 3 days. Tumor volume was calculated by the following equation: volume = (width2 × length)/2. The nude mice were euthanized by cervical dislocation 21 days later, and the tumors were collected, weighed, and stored at − 80°C for follow-up experiments. 2.10 Statistical analysis Statistical analysis was performed using SPSS25.0 software (IBM, SPSS, Armonk, New York, USA). Means, standard deviations, and unpaired Student's t test results were used to analyze the data. Relative gene expression was performed using the comparative 2 −∆∆Ct or 2 −∆Ct method. All experiments were performed with at least 3 independent biological replicates. P value < 0.05 was considered to be significant. 3. Results 3.1 LINC00963 is a super-enhancer-associated gene in GC We conducted an analysis of GEO data, specifically utilizing the downloaded GSE75595 dataset [ 20 ], for the characterization of super-enhancers in gastric cancer (GC) (Fig. 1 A). The analysis revealed the identification of 220, 420, 281, 123, and 72 super-enhancers in AGS cells, MKN45 cells, MKN1 cells, KATO Ⅲ cells, and SNU016 cells, respectively. Notably, super-enhancer target genes in AGS and KATO-Ⅲ cell lines were found to be involved in the tight junction signaling pathway, while MKN45 and SNU016 cells exhibited super-enhancer-associated genes primarily participating in the adherens junction signal pathway (Fig. 1 B). The Venn diagram analysis of the predicted target genes by the enhancer-super-enhancer indicated that ACTN4, KRT80, ZFP36, LINC00963, and MIR21 were common to all cell lines studied (The Venn diagram in Fig. 1 C). LINC00963 was successfully validated by RT-qPCR and selected as the focus for further investigation. The CCLE database was utilized to investigate the expression of LINC00963 in GC cell lines, revealing high expression level in these cell lines (Fig. 1 D). JQ1 was used to assess its effect on the function of enhancer-super-enhancer target genes in GC. The inhibitory effect on BRD4 in tumor cell super-enhancers was predominantly observed after 6 hours of exposure to 500 nM of JQ1 in GC cells. Consequently, GC cell lines were exposed to JQ1 at concentrations of 5, 50, 500, and 5000 nM for 6 hours. The LINC00963 gene demonstrated a decrease in expression at these concentrations throughout the specified duration. Remarkably, the most significant reduction in the level of LINC00963 expression in HGC-27 and MGC803 cell lines (Fig. 1 F) occurred at a JQ1 concentration of 500 nM. 3.2 LINC00963 is over-expressed in GC cell lines and peripheral blood, and it has alternative splice variants The specific primers were designed to amplify LINC00963 gene expression and its alternative splice variants. It was shown that through cloning and sequencing, the gene had multiple alternative splice variants that were not included in the NCBI LINC00963-V1(Banklt2765306 OR820605), LINC00963-V2(Banklt2765288 OR820604), and LINC00963-V3(Banklt2765324 OR820606) (Fig. 2 A-B). Sequencing results revealed the appearance of V1 with an additional 59 bases at the 5 'end of the second exon (Fig. 2 A). This addition was entirely consistent with the 59 bases at the 3’ end of the first intron, and the third exon was completely deleted. Variant 2 skips the third exon, while variant 3 skips both the second and third exons. The expression of LINC00963 and its newly discovered alternative splice variants were explored in gastric mucosal (GES-1) and GC cell lines (AGS, MKN45, HGC-27, and MGC803). In comparison to the GC HGC-27 cell line, LINC00963 gene expression was over tripled than the GES-1 cell line (Fig. 2 C). The level of LINC00963-V1 expression in the MGC803 cell line was not as high as the HGC-27 cell line, but was higher than gastric mucosal GES-1 cells (Fig. 2 C). The peripheral blood of 30 patients with GC and the peripheral blood of 30 healthy individuals at the time of physical examinations were collected to investigate the expression LINC00963, revealing that in the peripheral blood of GC patients, the expression was higher than in the peripheral blood of healthy individuals (Fig. 2 D-F). These results indicate that our newly discovered alternative splicing variants of LINC00963 are highly expressed in gastric cancer and may play an important biological functions. 3.3 LINC00963 promoted the EMT via the Wnt/β-catenin signal pathway in GC cell lines The relative expression of LINC00963-V1 was highest among the alternative splice variants of the LINC00963 gene (Fig. 2 B). The previous bioinformatics analysis showed that super-enhancer-associated genes were involved in cell metastasis (Fig. 1 B). The migration and invasion ability of HGC-27 and MGC803 cell lines were significantly weakened after knockdown of the LINC00963-V1 group compared to the si-NC group (Fig. 3 A, B, C, D). LINC00963 expression was knocked down by transient technology in GC cell lines HGC-27 and MGC803. Compared with the si-NC and the si-V1-a, si-V1-b, and si-V1-a + b groups, the number of apoptotic cells significantly increased after knocking down the LINC00963 in the HGC-27 and MGC803 cells (Fig. 3 E, F). The EMT process is crucial for gastric cancer cell metastasis. Research has shown that knockdown LINC00963 in the LINC00963-V1 group leads to increased expression of epithelial marker E-cadherin and decreased expression of mesenchymal markers N-cadherin and vimentin compared to the si-NC group. Moreover, expression of the EMT-related transcription factors SNAIL, ZEB1 and β-catenin were downregulated upon knockdown of LINC00963, indicating that high expression of LINC0963 promotes the occurrence and development of EMT (Fig. 4 A, B, C). 3.4 Suppression of LINC00963 inhibited GC cells growth in vivo In order to investigate the effect of LINC00963 on tumorigenicity in vivo, we inoculated sh-LINC00963 group tumor cells into the left groin of mice, and sh-NC group tumor cells into the right groin of BALB/c mice, establishing a mouse subcutaneous xenograft model. After 21 days of observation and feeding, euthanize the mice to remove the transplanted tumor. Figure 5 A demonstrates that the knockdown efficiency of sh-LINC00963-V1-a was higher than that of sh-LINC00963-V1-b. Throughout the experimental period, nude mice exhibited a steady increase in body weight, as depicted in Fig. 5 B. Cells transfected with lenti-shLINC00963 V1 displayed significantly reduced tumor volume and weight, as illustrated in Fig. 5 C and D. After euthanizing the mice, the results showed that the tumor weight of the LINC00963 knockdown group was significantly smaller than that of the control group, indicating that knockdown the expression of LINC00963 significantly inhibited tumor growth. As shown in the figure (Fig. 5 E, F). Then, RNA was extracted from the tumor tissue for detection, and it was found that the expression of LINC00963 in the sh-LINC00963 side of the tumor tissue was lower than that in the control side, as shown in Fig. 5 G. 4. Discussion As an important cis-acting element, super-enhancers are rich in transcription factors and cofactors, such as high-density BRD4[ 21 , 22 ], which greatly enhance gene expression[ 23 ]. Accumulating studies indicate that super-enhancers play a pivotal role in regulating target genes, fostering the malignant progression of tumors[ 24 – 27 ]. The LINC00963 gene is situated on human chromosome 9q34.11, and research indicates that its disorders are commonly involved in tumor invasion, metastasis, and disease deterioration. [ 28 – 30 ]. However, it remained unclear whether LINC00963 is associated with any super enhancers. In our study, we comprehensively characterized the super-enhancer landscape in GC by analyzing uploaded H3K27ac ChIP-seq data from GC cell lines (GSE75595). Subsequently, we identified LINC00963 as a super-enhancer-associated gene. It has been reported that transcripts sensitive to JQ1 are potential super-enhancer-related genes[ 31 ]. Our results demonstrated that low doses of JQ1 inhibited the transcription of LINC00963 in GC cell lines. These findings indirectly suggest that LINC00963 is regulated by super-enhancers. Furthermore, we observed high expression of LINC00963 in both GC cell lines and the peripheral blood of GC patients, implying a potential role for LINC00963 in the initiation and progression of GC. The types of proteins in humans have been shown to be far greater than the number of genes, due in part to multiple alternative splicing in genes translate proteins with different functions. We found multiple alternative splicing variants in GC, which may have a crucial role in the malignant progression of tumors. Our results showed that LINC00963 has at least three variants not included in the NCBI database, while the level of wild-type transcript (NR 038955.1) expression was very low. In several studies involving the alternative splicing of the LINC00963 gene, the newly discovered splicing variants is the deletion of the 5’end sequence[ 32 , 33 ]. Our research results showed that the LINC00963 alternative splicing was mainly caused by the 59 base increase in the 5’ end of the second exon and the deletion of the third exon, which may be due to the splicing variants produced by different malignant tumors. Notably, we did not perform a 5’ RACE test to verify whether there is a deletion of the RNA 5’ end of the LINC00963 gene. Because there are multiple splicing variants in LINC00963 and the LINC00963-V1 expression was the highest, it is still unknown whether the variable splicing event of LINC00963 was related to the mutation or deletion of some sites of LINC00963, which affected the post-transcriptional modification of RNA, this will be the focus of our further research. The LINC00963 expression was downregulated in both AGS and MKN45 cells. Although bioinformatics analysis showed that LINC00963 was the target gene of the super-enhancer in AGS and MKN45 cell lines, the reason for this result might be the high degree of differentiation between AGS and MKN45. The differences caused by the different sources of cancer cells when the cells were constructed may also be caused by some mutations in the continuous culture of cell lines, which may result in the low expression of LINC00963 or the weakened function of super-enhancers. In conclusion, the high expression of LINC00963 and its different splicing variants in peripheral blood indicates that LINC00963 may be a potential molecular marker of GC, which implys a new insight for LINC00963 studies. At present, accumulated studies have shown that EMT is closely related to the invasion and metastasis of tumors, and involves the signal transmission of Wnt/β-catenin, TGF-β, and other related pathways [ 30 , 34 – 37 ]. After the knockout of LINC00963-V1 in vitro , the invasion and migration ability of GC cells were greatly weakened, the EMT-related proteins changed, and the expression of β-catenin was decreased. As β-catenin is an important molecular marker in the Wnt/β-catenin signaling pathway, the Wnt/β-catenin signaling pathway has been associated with EMT in multiple tumor studies[ 17 , 38 ]. Numerous studies have shown that LINC00963 has an oncogene role in tumor occurrence and development, promoting the metastasis and invasion of cancer cells[ 39 – 41 ], which is consistent with our results. We also found that the LINC00963 gene fosters EMT of GC via the Wnt/β-catenin signaling pathway, thereby promoting the invasion and migration of GC cells. In summary, this study has unveiled multiple previously unreported alternative splicing variants of LINC00963 and identified the underlying mechanisms by which LINC00963 is involved in tumor invasion and migration relying on super-enhancer. Specifically, LINC00963 mediates the regulation of the Wnt/β-catenin signaling pathway through super-enhancer, promoting epithelial-mesenchymal transition of gastric cancer. These findings offer new insights into LINC00963 as a novel biomarker for GC metastasis and a potential therapeutic target in gastric cancer treatment. Declarations Institutional Review Board Statement: The collection of peripheral blood samples was authorized and approved by the Ethics Committee of the Affiliated Hospital of Guizhou Medical University (2020 [106]). The samples were sourced with the consent of the patients and family members. The study was approved by the Animal Care Welfare Committee of Guizhou Medical University (2200732). Author Contributions: HD, YJ, TT-X, CF-S and HH designed the study. HD and TT-X analyzed and discussed the data. HD, TT-X and YX wrote the manuscript. YX, DH, and TT-X participated in the animal experiment. WS-X and FH-D performed experiments and examined the data. SX-W, HY-L, SQ-C and BX-L coordinated sample collection and processing and discussed the data. HH and YX critically reviewed and edited the manuscript. All authors have read and approved the article. Funding: This study was supported by research grants from the National Natural Science Foundation of China to H.H. (82060442) and the Foundation of the Education Department of Guizhou Province to Y.X. (no. [2022]214). This work was partly funded by the China Scholarship Council (No. 202008520053 to Y.X.). Conflicts of Interest: The authors declare no conflict of interest. References Y. Tarumoto, S. Lin, J. Wang, et al., Salt-inducible kinase inhibition suppresses acute myeloid leukemia progression in vivo, Blood 135 (2020) 56–70. 10.1182/blood.2019001576 . Y.-X. Zhang, K. Yang, Significance of nodal dissection and nodal positivity in gastric cancer, Transl Gastroenterol Hepatol 5 (2020) 17. 10.21037/tgh.2019.09.13 . S.E. Oh, J.Y. An, M.-G. 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Li, et al., Transcriptional super-enhancers control cancer stemness and metastasis genes in squamous cell carcinoma, Nat Commun 12 (2021) 3974. 10.1038/s41467-021-24137-1 . Z. Ghaemi, S.J. Mowla, B.M. Soltani, Novel splice variants of LINC00963 suppress colorectal cancer cell proliferation via miR-10a/miR-143/miR-217/miR-512-mediated regulation of PI3K/AKT and Wnt/β-catenin signaling pathways, Biochim Biophys Acta Gene Regul Mech 1866 (2023) 194921. 10.1016/j.bbagrm.2023.194921 . T. Yu, Y. Zhao, Z. Hu, et al., MetaLnc9 Facilitates Lung Cancer Metastasis via a PGK1-Activated AKT/mTOR Pathway, Cancer Res 77 (2017) 5782–5794. 10.1158/0008-5472.CAN-17-0671 . M. Buccarelli, V. Lulli, A. Giuliani, et al., Deregulated expression of the imprinted DLK1-DIO3 region in glioblastoma stemlike cells: tumor suppressor role of lncRNA MEG3, Neuro Oncol 22 (2020) 1771–1784. 10.1093/neuonc/noaa127 . S. Chen, M. Yang, C. Wang, et al., Forkhead box D1 promotes EMT and chemoresistance by upregulating lncRNA CYTOR in oral squamous cell carcinoma, Cancer Lett 503 (2021) 43–53. 10.1016/j.canlet.2020.11.046 . T. Jiang, W. Gao, S. Lin, et al., FNDC1 Promotes the Invasiveness of Gastric Cancer via Wnt/β-Catenin Signaling Pathway and Correlates With Peritoneal Metastasis and Prognosis, Front Oncol 10 (2020) 590492. 10.3389/fonc.2020.590492 . M. Zhu, N. Zhang, X. Lu, et al., Negative Regulation of Kruppel-Like Factor 4 on microRNA-106a at Upstream Transcriptional Level and the Role in Gastric Cancer Metastasis, Dig Dis Sci 63 (2018) 2604–2616. 10.1007/s10620-018-5143-z . G. Jiang, W. Deng, Y. Liu, et al., General mechanism of JQ1 in inhibiting various types of cancer, Mol Med Rep 21 (2020) 1021–1034. 10.3892/mmr.2020.10927 . R. Hu, B. Xu, J. Ma, et al., LINC00963 promotes the malignancy and metastasis of lung adenocarcinoma by stabilizing Zeb1 and exosomes-induced M2 macrophage polarization, Mol Med 29 (2023) 1. 10.1186/s10020-022-00598-y . F. Sun, K. Wu, Z. Yao, et al., Long noncoding RNA LINC00963 induces NOP2 expression by sponging tumor suppressor miR-542-3p to promote metastasis in prostate cancer, Aging (Albany NY) 12 (2020) 11500–11516. 10.18632/aging.103236 . J. Ye, J. Liu, T. Tang, et al., LINC00963 affects the development of colorectal cancer via MiR-532-3p/HMGA2 axis, Cancer Cell Int 21 (2021) 87. 10.1186/s12935-020-01706-w . Additional Declarations No competing interests reported. Supplementary Files SupplementaryData.docx Cite Share Download PDF Status: Posted Version 1 posted 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-4020742","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277935638,"identity":"75fb593d-2781-45ac-8119-2f9acced95d7","order_by":0,"name":"Hong Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDCCAzwg0gbC4SFBSxrpWg6ToIXvRu7BxwW/ztutbT/A+OBtG4O8OSEtkjfyko1n9t1O3nYmgdlwbhuD4c4GAloMbuSYSfP23E42u8HAJs3bxpBgcIA4LedAWth/E6+F58cBO5AtzERpkTzzxtiYtyE5wexMYrPknHMShhsIaeE7nmP4mOePnb3Z8cMHP7wps5EnaAsYMLYxJDYwMDYAmRLEqAeBPwz2xCodBaNgFIyCEQgAqHBBxTLHk0kAAAAASUVORK5CYII=","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hong","middleName":"","lastName":"Du","suffix":""},{"id":277935639,"identity":"0419cf87-d748-4923-bdfe-dc834ebbf356","order_by":1,"name":"Tingting Xiang","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Xiang","suffix":""},{"id":277935640,"identity":"6cced220-6c97-4b56-b148-39601fba68ba","order_by":2,"name":"Ying Xia","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Xia","suffix":""},{"id":277935641,"identity":"e73ffbad-40c4-470d-af98-47b7d1cabaeb","order_by":3,"name":"Yong Jin","email":"","orcid":"","institution":"The Second People’s Hospital of Guizhou Province","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Jin","suffix":""},{"id":277935642,"identity":"a4591727-fc0f-4774-9365-545e569f94e4","order_by":4,"name":"Fahua Deng","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fahua","middleName":"","lastName":"Deng","suffix":""},{"id":277935643,"identity":"edb50f97-f0e5-431e-b900-8ed266dbd1c1","order_by":5,"name":"Wansong Xia","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wansong","middleName":"","lastName":"Xia","suffix":""},{"id":277935644,"identity":"a9b0dd2c-27ac-47c7-bd7e-f5a2c8dfd75a","order_by":6,"name":"Hongyu Li","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Li","suffix":""},{"id":277935645,"identity":"41b20bae-3b91-443d-8680-8c59881dbfbb","order_by":7,"name":"Shuqiang Cheng","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuqiang","middleName":"","lastName":"Cheng","suffix":""},{"id":277935646,"identity":"034fd190-1cab-44da-8534-4831588a211e","order_by":8,"name":"Bingxue Lan","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bingxue","middleName":"","lastName":"Lan","suffix":""},{"id":277935647,"identity":"1ad4f493-8b46-4a38-bd75-8416136de4ad","order_by":9,"name":"Sixi Wei","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sixi","middleName":"","lastName":"Wei","suffix":""},{"id":277935648,"identity":"7ad282ed-c52b-4ce2-984d-5bb92a856c92","order_by":10,"name":"Cunfeng Song","email":"","orcid":"","institution":"National Engineering Center for Nanotechnology","correspondingAuthor":false,"prefix":"","firstName":"Cunfeng","middleName":"","lastName":"Song","suffix":""},{"id":277935649,"identity":"d3aef917-cf78-4a95-ab57-cf8742dd2553","order_by":11,"name":"Hai Huang","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hai","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-03-06 11:48:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4020742/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4020742/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52595565,"identity":"bb5e8751-f472-408d-a9d9-2721bfdfa524","added_by":"auto","created_at":"2024-03-13 11:46:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147042,"visible":true,"origin":"","legend":"\u003cp\u003eGEO database download data (GSE75595) analysis and identification of gastric cancer cell line super-enhancer and prediction of target genes. A. H3K27ac to define the active enhancers in the whole cell genome, sorted by H3K27ac enrichment intensity, and found the point with a slope of 1 as the threshold value of normal- and super-enhancers. B. KEGG analyzed the super-enhancer target gene enrichment signal pathways corresponding to each cell, and it is clear that the main signal regulation pathways in the five cell lines are tight and adherens junctions. C. A Venn diagram was made for super-enhancer target genes. Five cell lines and five public super-enhancer target genes were found, including ACTN4, KRT80, ZFP36, LINC00963, and MIR21. D. CCLE database analysis shows that the LINC00963 gene is highly expressed in gastric cancer cell lines. E-F. The inhibitory effect of different doses of the JQ1 inhibitor on HGC-27(E) and MGC803(F) cell line LINC00963 different alternative spliceosomes (6 h). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05,**\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01,***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-4020742/v1/d79351f1de700e1b2d99b31a.png"},{"id":52595621,"identity":"24b6af14-a83e-4ec0-a780-b12504c7ecf6","added_by":"auto","created_at":"2024-03-13 11:47:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":870996,"visible":true,"origin":"","legend":"\u003cp\u003eLINC00963 discovery of new alternative spliceosomes and expression analysis. A: LINC00963 The structure of the newly discovered variable splicosome, the number of exons and bases and introns, and the binding sites of all primers, the siRNA design is located within the red box. B: The theoretical length of LINC00963-F503/R1557 is 1054bp, separated by 2% agarose gel and cloned, sequenced, and analyzed to obtain three unreported alternative spliceosomes of the LINC00963 gene. Among them, the second exon of V1 had 59 more than the 5’ end. Bases. C: The relative expression analysis of the total alternative spliceosome (ALL) of the LINC00963 gene and the newly discovered alternative spliceosome in gastric cancer cell lines with the GES-1 cell line as a control. D: The expression of all alternative spliceosomes of the LINC00963 gene in the peripheral blood of gastric cancer patients and healthy individuals at the time of a physical examination. E: The expression of LINC00963-V1 in the peripheral blood of gastric cancer patients and healthy individuals at the time of a physical examination. F: The expression of LINC00963-V2 in the peripheral blood of gastric cancer patients and healthy individuals at the time of physical examination. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig21.png","url":"https://assets-eu.researchsquare.com/files/rs-4020742/v1/372d5a3827538ac389dab164.png"},{"id":52595567,"identity":"6c32cd00-e2e6-4a59-ade5-0b6e51b2c01c","added_by":"auto","created_at":"2024-03-13 11:46:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":305640,"visible":true,"origin":"","legend":"\u003cp\u003eLINC00963-V1 knockdown to detect the invasion and migration ability of HGC-27 and MGC803 cells. A-B: siRNA with FAM fluorescence was transfected into the cells to detect the cell transfection efficiency at 6 h. The RNA was extracted 48 h after transfection to detect the knockdown efficiency; the knockdown efficiency was \u0026gt;80%. C-E: 48 h after transfection of cell line, detection of HGC-27 and MGC803 cell invasion and migration, showed that the ability of cell invasion and migration decreased after LINC00963-V1 knockdown compared with the si-NC group. F. Flow cytometry result of HGC-27, MGC803si-NC, si-V1-a, si-V1-b, and si-V1-a+b apoptosis. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig31.png","url":"https://assets-eu.researchsquare.com/files/rs-4020742/v1/538cce2c3efbb59df518d2c3.png"},{"id":52596056,"identity":"8e0903c1-5950-4ad9-a0fa-f9fd0292363c","added_by":"auto","created_at":"2024-03-13 11:54:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":870996,"visible":true,"origin":"","legend":"","description":"","filename":"Fig21.png","url":"https://assets-eu.researchsquare.com/files/rs-4020742/v1/3d59f88cad299b2fae63be7b.png"},{"id":52595562,"identity":"ee3bcefa-19ac-413d-8f1b-da03189194c4","added_by":"auto","created_at":"2024-03-13 11:46:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1473820,"visible":true,"origin":"","legend":"\u003cp\u003eLINC00963-V1 knockdown attenuated EMT in GC HGC-27 and MGC803 cells. A: The EMT-associated proteins in HGC-27 and MGC803 cells were detected by western blot assay after si-V1-a, si-V1-b, and si-NC transfection compared with the si-NC group. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig41.png","url":"https://assets-eu.researchsquare.com/files/rs-4020742/v1/3b41929c1f61a80d79cd85b9.png"},{"id":52595563,"identity":"7df8a714-ed73-4a7d-b81e-df0874cd1a6b","added_by":"auto","created_at":"2024-03-13 11:46:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1812177,"visible":true,"origin":"","legend":"\u003cp\u003eStable knockdown of LINC00963 expression inhibited the invasion, migration, and tumor growth of the gastric cancer cell line, HGC-27. A. Lentivirus vector plasmid stably knockdown of LINC00963 expression. B-D. The tumorigenesis experiment, B) Average weight of nude mice, C) Photo of tumor volume \u003cem\u003ein vivo\u003c/em\u003e, the left side was sh-NC, and the right side was sh-LINC00963-V1-a. D. The curves of tumor volumes. E-F. Pictures and weight statistics of tumors \u003cem\u003ein vitro\u003c/em\u003e. G. The expression of LINC00963 in tumor tissue. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig51.png","url":"https://assets-eu.researchsquare.com/files/rs-4020742/v1/284ee35ca6e0c43de76ca3dc.png"},{"id":53281731,"identity":"cb837fe3-883d-4c9b-9f9e-af31d18fea19","added_by":"auto","created_at":"2024-03-22 20:01:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2971297,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4020742/v1/ea1f16c8-f496-46ff-ab5f-64b57c960ae8.pdf"},{"id":52537863,"identity":"41a30885-d63a-4b9f-aebb-551cb33d0e4a","added_by":"auto","created_at":"2024-03-12 16:48:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":805834,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-4020742/v1/e5d9614c6eb9f11fa2d6b895.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Super-enhancer-associated LINC00963 promotes epithelial-mesenchymal transition in gastric cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn clinical practice, gastric cancer (GC) is a common malignancy with high morbidity. According to the Global Cancer Report, in 2020, GC ranks 5th in amount of new cancer cases (5.6% of 19.3\u0026nbsp;million), and 4th in cancer-related deaths (7.7% of 10\u0026nbsp;million) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Invasion and metastasis are the main reasons for the low survival rate and poor prognosis of GC patients[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As a consequence, it is important to elucidate the molecular mechanisms of GC invasion and metastasis in order to identify effective GC prognostic molecular markers and corresponding therapeutic targets.\u003c/p\u003e \u003cp\u003eA super-enhancer (SE) is a genomic region in mammals, often spanning an average size of up to 30 kb, that comprises multiple enhancers[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These enhancers work collectively to recruit core regulatory circuitry transcription factors (TFs), thus becoming an important cause of the mediated transcriptional dysregulation which is observed in human cancers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Super-enhancers regulate the abnormal expression of specific genes to promote the malignant process of tumors[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] in pancreatic cancer[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], colorectal cancer[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], Ewing sarcoma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and GC[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It has also been reported that super-enhancers are more sensitive to drugs, such as JQ1 and THZ1, than typical enhancers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Low doses of JQ1 can interfere with super-enhancer function, thereby inhibiting the abnormal expression of the related downstream target genes, most of which are specific genes that can identify cells. As a result, the occurrence and development of tumors were suppressed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].. Enhancer-mediated genes have been reported to promote tissue invasion, such as myocyte enhancer factor 2C (MEF2C)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Our previous research has identified several lncRNAs associated with the occurrence, development, and metastasis of gastric cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We speculate that since SE can modulate specific genes expression to promote the process of tumor development, it is also conceivable that SEs may similarly regulate the lncRNAs expression, resulting in the promotion of malignant progression. However, there have been few reports on super-enhancer-associated genes regulating invasion and migration in GC to date.\u003c/p\u003e \u003cp\u003eThis work investigated the function and its underlying mechanisms of a SE-associated gene LINC00963 in promoting GC progression with in vivo, in vitro experiments and clinical cases. The underlying molecular mechanisms were also elucidated that SE-associated LINC00963 promotes GC invasion and migration through the Wnt/β-catenin pathway. The results indicate that LINC00963 is a potential marker for labeling the metastasis of GC.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cells cultivation\u003c/h2\u003e \u003cp\u003eGastric cancer cell lines (AGS, MKN45, HGC-27, and MGC803) and an immortalized gastric epithelial cell line (GES-1) were purchased from the National Infrastructure of Cell Line Resource of China (Beijing, China). All cell lines were cultured in RPMI-1640 medium (Gibco, Carlsbad, California, USA) containing 10% fetal bovine serum (Lonza Science SRL, Basel, Switzerland) and 1% penicillin-streptomycin (Invitrogen, Shanghai, China) and incubated under 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Short tandem repeats (STRs) of cell lines and \u003cem\u003eMycoplasma\u003c/em\u003e contamination were checked regularly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 human GC samples\u003c/h2\u003e \u003cp\u003eThe peripheral blood of 30 patients with GC and the peripheral blood of 30 healthy individuals at the time of physical examinations were collected 2mL from GC patients and healthy individuals in the Affiliated Hospital of Guizhou Medical University (Guizhou, China). The blood samples were labeled and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. The collection of peripheral blood samples was authorized and approved by the Ethics Committee of the Affiliated Hospital of Guizhou Medical University (2020 [106]). Blood samples are taken with the verbal consent of the patient and family.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Super-enhancer data analysis of GC cell lines\u003c/h2\u003e \u003cp\u003eThe GC cell line enhancer-super-enhancer data is available through GEO under accession number GSE75595 (Accessed in 2018). The GC cell line enhancer-super-enhancer data is available through GEO under accession number GSE75595 (Accessed in 2019). Gene Ontology (GO) enrichment analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to describe the super-enhancer target gene function. To predict enhancer-super-enhancer target gene sets, we performed biological pathway enrichment analysis based on the KEGG database, referencing the comprehensive data analysis of excellent enhancers in the previous work reported by Whyte WA and Wang J[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The Cancer Cell Line Encyclopedia (CCLE) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.broadinstitute.org/ccle\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.broadinstitute.org/ccle\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to comprehensively analyze the gene expression of GC cell lines, particularly the LINC00963 gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RNA extraction and RT- qPCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from peripheral blood and GC cell line using the UNlQ-10 column Trizol RNA extraction kit (B511321-0100; Sangon Biotech Co., Ltd., Shanghai, China). Expression of the target genes was quantified with RT-qPCR kits (Takara, Cat. DRR820A), normalized to the housekeeping gene HPRT. The relative gene expression levels were determined by the comparative threshold cycle (2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e) method. The primer sequences of RT-qPCR are listed in \u003cb\u003eSupplementary Table\u0026nbsp;1.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Small interfering RNA (siRNA) transfection and lentivirus vector construction\u003c/h2\u003e \u003cp\u003eThe full-length sequences of LINC00963 (NR_038955.1) and the newly discovered LINC00963-V1 were obtained according to the NCBI database. Specific siRNAs for LINC00963-V1 were designed using siRNA technology, and then synthesized by Shanghai Gene Pharma Co., Ltd. (Shanghai, China). The siRNA sequences are listed in \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e. Lipofectamine\u0026reg; RNAi MAX reagent (Invitrogen, Shanghai, China) was used to transfect siRNA. The cell lines of stable LINC00963 knockdown were established by lentivirus-mediated delivery of LINC00963-specific shRNA (sh-LINC00963). Lentivirus packaging kit GM easy\u0026trade; (Genomeditech, Shanghai, China) was used to synthesize lentivirus. Lentivirus vector is pPLK plasmid sh-LINC00963 (public protein/plasmid library). The shRNA sequences are listed in \u003cb\u003eSupplement Table\u0026nbsp;3\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Migration and invasion assays\u003c/h2\u003e \u003cp\u003eTranswell determination using 8.0 \u0026micro;m pore size Transwell permeable carrier (Corning, New York, USA) was used. The number of cells used for cell migration and invasion determination was 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e and 6 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, respectively. After the experiment, they were fixed in methanol and stained with crystal violet solution for photography[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Apoptosis assay\u003c/h2\u003e \u003cp\u003eApoptotic cells were assayed by the cell apoptosis assay kit (Beyotime Biotechnology, Shanghai, China). Briefly, HGC-27 or MGC803 cells were seeded into 6-well plates and transfected with a negative control siRNA (si-NC) or LINC00963 siRNA. After a 72-h transfection, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were collected from each group and washed twice with cold PBS. Binding buffer (500 \u0026micro;l) was added to resuspend the cells in each sample tube. Annexin V-TITC (5 \u0026micro;l) was added and gently mixed, then incubated at room temperature for 15 min in the dark. PI (10 \u0026micro;l) was added and incubated on ice for 5 min in the dark and detected with flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Immunoblotting assay\u003c/h2\u003e \u003cp\u003eImmunoblotting assay was performed according to our previous studies[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The following antibodies are used: GAPDH (Bioworld, Nanjing, China; 1:5000); E-cadherin (ab40772, Abcam, Boston, Massachusetts, USA; 1:1000); N-cadherin (ab76011, Abcam; 1:1000); vimentin (ET1610-39, Huabio, Hangzhou, China; 1:20000); MMP7 (ab205525, Abcam; 1:1000); β-catenin (ab32572, Abcam; 1:400); Zeb1 (WL03489, Wanleibio, Shenyang, China; 1:2000); Snail (WL01863, Wanleibio; 1:2000); and horseradish peroxidase-labeled goat anti-rabbit IgG (Multi Sciences, Hangzhou, China; 1:5000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 In vivo experiments\u003c/h2\u003e \u003cp\u003eFemale BALB/c nude mice (Age, 5weeks; Weight, 20\u0026thinsp;\u0026plusmn;\u0026thinsp;3g) were purchased from the SLRC Laboratory Animal Center (Shanghai, China). A dose 1\u0026times;106 HGC-27 cells from sh-LINC00963 were subcutaneously injected into the right inguinal region and sh-NC were injected into the left inguinal region of BALB/c nude mice. A total of 5 mice. The tumor volume and body weight were monitored every 3 days. Tumor volume was calculated by the following equation: volume = (width2 \u0026times; length)/2. The nude mice were euthanized by cervical dislocation 21 days later, and the tumors were collected, weighed, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for follow-up experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS25.0 software (IBM, SPSS, Armonk, New York, USA). Means, standard deviations, and unpaired Student's t test results were used to analyze the data. Relative gene expression was performed using the comparative 2 \u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e or 2 \u003csup\u003e\u0026minus;∆Ct\u003c/sup\u003e method.\u003c/p\u003e \u003cp\u003eAll experiments were performed with at least 3 independent biological replicates. \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 LINC00963 is a super-enhancer-associated gene in GC\u003c/h2\u003e \u003cp\u003eWe conducted an analysis of GEO data, specifically utilizing the downloaded GSE75595 dataset [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], for the characterization of super-enhancers in gastric cancer (GC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The analysis revealed the identification of 220, 420, 281, 123, and 72 super-enhancers in AGS cells, MKN45 cells, MKN1 cells, KATO Ⅲ cells, and SNU016 cells, respectively. Notably, super-enhancer target genes in AGS and KATO-Ⅲ cell lines were found to be involved in the tight junction signaling pathway, while MKN45 and SNU016 cells exhibited super-enhancer-associated genes primarily participating in the adherens junction signal pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The Venn diagram analysis of the predicted target genes by the enhancer-super-enhancer indicated that ACTN4, KRT80, ZFP36, LINC00963, and MIR21 were common to all cell lines studied (The Venn diagram in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). LINC00963 was successfully validated by RT-qPCR and selected as the focus for further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CCLE database was utilized to investigate the expression of LINC00963 in GC cell lines, revealing high expression level in these cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). JQ1 was used to assess its effect on the function of enhancer-super-enhancer target genes in GC. The inhibitory effect on BRD4 in tumor cell super-enhancers was predominantly observed after 6 hours of exposure to 500 nM of JQ1 in GC cells. Consequently, GC cell lines were exposed to JQ1 at concentrations of 5, 50, 500, and 5000 nM for 6 hours. The LINC00963 gene demonstrated a decrease in expression at these concentrations throughout the specified duration. Remarkably, the most significant reduction in the level of LINC00963 expression in HGC-27 and MGC803 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) occurred at a JQ1 concentration of 500 nM.\u003c/p\u003e \u003ch2\u003e3.2 LINC00963 is over-expressed in GC cell lines and peripheral blood, and it has alternative splice variants\u003c/h2\u003e \u003cp\u003eThe specific primers were designed to amplify LINC00963 gene expression and its alternative splice variants. It was shown that through cloning and sequencing, the gene had multiple alternative splice variants that were not included in the NCBI LINC00963-V1(Banklt2765306 OR820605), LINC00963-V2(Banklt2765288 OR820604), and LINC00963-V3(Banklt2765324 OR820606) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Sequencing results revealed the appearance of V1 with an additional 59 bases at the 5 'end of the second exon (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This addition was entirely consistent with the 59 bases at the 3\u0026rsquo; end of the first intron, and the third exon was completely deleted. Variant 2 skips the third exon, while variant 3 skips both the second and third exons. The expression of LINC00963 and its newly discovered alternative splice variants were explored in gastric mucosal (GES-1) and GC cell lines (AGS, MKN45, HGC-27, and MGC803). In comparison to the GC HGC-27 cell line, LINC00963 gene expression was over tripled than the GES-1 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The level of LINC00963-V1 expression in the MGC803 cell line was not as high as the HGC-27 cell line, but was higher than gastric mucosal GES-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The peripheral blood of 30 patients with GC and the peripheral blood of 30 healthy individuals at the time of physical examinations were collected to investigate the expression LINC00963, revealing that in the peripheral blood of GC patients, the expression was higher than in the peripheral blood of healthy individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). These results indicate that our newly discovered alternative splicing variants of LINC00963 are highly expressed in gastric cancer and may play an important biological functions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 LINC00963 promoted the EMT via the Wnt/β-catenin signal pathway in GC cell lines\u003c/h2\u003e \u003cp\u003eThe relative expression of LINC00963-V1 was highest among the alternative splice variants of the LINC00963 gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The previous bioinformatics analysis showed that super-enhancer-associated genes were involved in cell metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The migration and invasion ability of HGC-27 and MGC803 cell lines were significantly weakened after knockdown of the LINC00963-V1 group compared to the si-NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, C, D). LINC00963 expression was knocked down by transient technology in GC cell lines HGC-27 and MGC803. Compared with the si-NC and the si-V1-a, si-V1-b, and si-V1-a\u0026thinsp;+\u0026thinsp;b groups, the number of apoptotic cells significantly increased after knocking down the LINC00963 in the HGC-27 and MGC803 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe EMT process is crucial for gastric cancer cell metastasis. Research has shown that knockdown LINC00963 in the LINC00963-V1 group leads to increased expression of epithelial marker E-cadherin and decreased expression of mesenchymal markers N-cadherin and vimentin compared to the si-NC group. Moreover, expression of the EMT-related transcription factors SNAIL, ZEB1 and β-catenin were downregulated upon knockdown of LINC00963, indicating that high expression of LINC0963 promotes the occurrence and development of EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Suppression of LINC00963 inhibited GC cells growth in vivo\u003c/h2\u003e \u003cp\u003eIn order to investigate the effect of LINC00963 on tumorigenicity in vivo, we inoculated sh-LINC00963 group tumor cells into the left groin of mice, and sh-NC group tumor cells into the right groin of BALB/c mice, establishing a mouse subcutaneous xenograft model. After 21 days of observation and feeding, euthanize the mice to remove the transplanted tumor. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA demonstrates that the knockdown efficiency of sh-LINC00963-V1-a was higher than that of sh-LINC00963-V1-b. Throughout the experimental period, nude mice exhibited a steady increase in body weight, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. Cells transfected with lenti-shLINC00963 V1 displayed significantly reduced tumor volume and weight, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D. After euthanizing the mice, the results showed that the tumor weight of the LINC00963 knockdown group was significantly smaller than that of the control group, indicating that knockdown the expression of LINC00963 significantly inhibited tumor growth. As shown in the figure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Then, RNA was extracted from the tumor tissue for detection, and it was found that the expression of LINC00963 in the sh-LINC00963 side of the tumor tissue was lower than that in the control side, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAs an important cis-acting element, super-enhancers are rich in transcription factors and cofactors, such as high-density BRD4[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which greatly enhance gene expression[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Accumulating studies indicate that super-enhancers play a pivotal role in regulating target genes, fostering the malignant progression of tumors[\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The LINC00963 gene is situated on human chromosome 9q34.11, and research indicates that its disorders are commonly involved in tumor invasion, metastasis, and disease deterioration. [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, it remained unclear whether LINC00963 is associated with any super enhancers. In our study, we comprehensively characterized the super-enhancer landscape in GC by analyzing uploaded H3K27ac ChIP-seq data from GC cell lines (GSE75595). Subsequently, we identified LINC00963 as a super-enhancer-associated gene. It has been reported that transcripts sensitive to JQ1 are potential super-enhancer-related genes[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our results demonstrated that low doses of JQ1 inhibited the transcription of LINC00963 in GC cell lines. These findings indirectly suggest that LINC00963 is regulated by super-enhancers. Furthermore, we observed high expression of LINC00963 in both GC cell lines and the peripheral blood of GC patients, implying a potential role for LINC00963 in the initiation and progression of GC.\u003c/p\u003e \u003cp\u003eThe types of proteins in humans have been shown to be far greater than the number of genes, due in part to multiple alternative splicing in genes translate proteins with different functions. We found multiple alternative splicing variants in GC, which may have a crucial role in the malignant progression of tumors. Our results showed that LINC00963 has at least three variants not included in the NCBI database, while the level of wild-type transcript (NR 038955.1) expression was very low. In several studies involving the alternative splicing of the LINC00963 gene, the newly discovered splicing variants is the deletion of the 5\u0026rsquo;end sequence[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our research results showed that the LINC00963 alternative splicing was mainly caused by the 59 base increase in the 5\u0026rsquo; end of the second exon and the deletion of the third exon, which may be due to the splicing variants produced by different malignant tumors. Notably, we did not perform a 5\u0026rsquo; RACE test to verify whether there is a deletion of the RNA 5\u0026rsquo; end of the LINC00963 gene. Because there are multiple splicing variants in LINC00963 and the LINC00963-V1 expression was the highest, it is still unknown whether the variable splicing event of LINC00963 was related to the mutation or deletion of some sites of LINC00963, which affected the post-transcriptional modification of RNA, this will be the focus of our further research. The LINC00963 expression was downregulated in both AGS and MKN45 cells. Although bioinformatics analysis showed that LINC00963 was the target gene of the super-enhancer in AGS and MKN45 cell lines, the reason for this result might be the high degree of differentiation between AGS and MKN45. The differences caused by the different sources of cancer cells when the cells were constructed may also be caused by some mutations in the continuous culture of cell lines, which may result in the low expression of LINC00963 or the weakened function of super-enhancers. In conclusion, the high expression of LINC00963 and its different splicing variants in peripheral blood indicates that LINC00963 may be a potential molecular marker of GC, which implys a new insight for LINC00963 studies.\u003c/p\u003e \u003cp\u003eAt present, accumulated studies have shown that EMT is closely related to the invasion and metastasis of tumors, and involves the signal transmission of Wnt/β-catenin, TGF-β, and other related pathways [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. After the knockout of LINC00963-V1 \u003cem\u003ein vitro\u003c/em\u003e, the invasion and migration ability of GC cells were greatly weakened, the EMT-related proteins changed, and the expression of β-catenin was decreased. As β-catenin is an important molecular marker in the Wnt/β-catenin signaling pathway, the Wnt/β-catenin signaling pathway has been associated with EMT in multiple tumor studies[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Numerous studies have shown that LINC00963 has an oncogene role in tumor occurrence and development, promoting the metastasis and invasion of cancer cells[\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which is consistent with our results. We also found that the LINC00963 gene fosters EMT of GC via the Wnt/β-catenin signaling pathway, thereby promoting the invasion and migration of GC cells.\u003c/p\u003e \u003cp\u003eIn summary, this study has unveiled multiple previously unreported alternative splicing variants of LINC00963 and identified the underlying mechanisms by which LINC00963 is involved in tumor invasion and migration relying on super-enhancer. Specifically, LINC00963 mediates the regulation of the Wnt/β-catenin signaling pathway through super-enhancer, promoting epithelial-mesenchymal transition of gastric cancer. These findings offer new insights into LINC00963 as a novel biomarker for GC metastasis and a potential therapeutic target in gastric cancer treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe collection of peripheral blood samples was authorized and approved by the Ethics Committee of the Affiliated Hospital of Guizhou Medical University (2020 [106]). The samples were sourced with the consent of the patients and family members. The study was approved by the Animal Care Welfare Committee of Guizhou Medical University (2200732).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e HD, YJ, TT-X, CF-S and HH designed the study. HD and TT-X analyzed and discussed the data. HD, TT-X and YX wrote the manuscript. YX, DH, and TT-X participated in the animal experiment. WS-X and FH-D performed experiments and examined the data. SX-W, HY-L, SQ-C and BX-L coordinated sample collection and processing and discussed the data. HH and YX critically reviewed and edited the manuscript. All authors have read and approved the article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was supported by research grants from the National Natural Science Foundation of China to H.H. (82060442) and the Foundation of the Education Department of Guizhou Province to Y.X. (no. [2022]214). This work was partly funded by the China Scholarship Council (No. 202008520053 to Y.X.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eY. Tarumoto, S. Lin, J. 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Tang, et al., LINC00963 affects the development of colorectal cancer via MiR-532-3p/HMGA2 axis, Cancer Cell Int 21 (2021) 87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12935-020-01706-w\u003c/span\u003e\u003cspan address=\"10.1186/s12935-020-01706-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gastric cancer, Super enhancer, LINC00963, Epithelial-mesenchymal transition","lastPublishedDoi":"10.21203/rs.3.rs-4020742/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4020742/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn clinical practice, gastric cancer (GC) is a common malignancy with high morbidity. Accumulating research has revealed that lncRNAs are involved in the development and metastasis of tumor tissues in multiple cancers. As reported, LINC00963, a typical lncRNA is aberrantly expressed in gastric cancer. However, the underlying mechanisms of super-enhancers mediating remain unclear.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eThe GC cell line enhancer-super-enhancer data were downloaded and analyzed from the NCBI database (GSE75595). Combined RT-qPCR and Sanger sequencing were employed to identify three variants of LINC00963 in gastric cell lines and peripheral blood samples from gastric cancer patients. The gene expression was detected through RT-qPCR, and their encoded protein expression levels were mesured by western blot. Transwell assayswere applied to assess the cell invasion and migration, while the apoptosis rate was tested by flow cytometry. A xenograft model was applied to simulate the tumor development process, during which the effect of LINC00963 on promoting tumorigenesis were investigated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAnalysis of the GC cell line enhancer-super-enhancer data revealed a high expression of LINC00963 driven by super-enhancer. The variant 1 and variant 2 of LINC00963 exhibited high expression in GC cell line and the peripheral blood of gastric cancers. After down-regulated variant 1 of LINC00963, the results showed a significant increase in cell apoptosis rate. LINC00963 expression in GC cell line reduced upon exposure to a low dose of the bromodomain and extra-terminal inhibitor, JQ1, which resulted in a decrease of the protein levels of β-catenin and ZEB1. As a result, the protein expression levels of several marker proteins related to epithelial-mesenchymal transition (EMT), such as Vimentin, N-cadherin and Snail were observed to decrease, which may lead to an inhibition on GC cells metastasis, thereby suppressing tumor growth.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study demonstrated that the Super-enhancer-associated LINC00963 is via a Wnt/β-catenin pathway that promotes EMT and tumor metastasis in gastric cancer.\u003c/p\u003e","manuscriptTitle":"Super-enhancer-associated LINC00963 promotes epithelial-mesenchymal transition in gastric cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-12 16:48:47","doi":"10.21203/rs.3.rs-4020742/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"132164e5-367d-4e32-918f-6996a0ef06ef","owner":[],"postedDate":"March 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-22T20:00:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-12 16:48:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4020742","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4020742","identity":"rs-4020742","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}