LncRNA DDX11-AS1 promotes breast cancer progression via targeting the miR-30c-5p/MTDH axis

LncRNA DDX11-AS1 promotes breast cancer progression via targeting the miR-30c-5p/MTDH axis | 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 LncRNA DDX11-AS1 promotes breast cancer progression via targeting the miR-30c-5p/MTDH axis Yanting Li, Mengsi Zhou, Liu Yang, Shuo Liu, Lixian Yang, Bin Xu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3822928/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 Introduction Long non-coding RNAs (lncRNAs) serve a significant role in the occurrence and development of malignant tumors. However, the roles of lncRNAs in breast cancer (BC) remain largely unknown. Therefore, the current study aimed to investigate the effect of lncRNA DDX11-AS1 on BC progression. Methods Bioinformatics analysis using public microarray revealed that DDX11-AS1 was upregulated in BC. In addition, the effect of DDX11-AS1 on the prognosis of patients with BC was evaluated by clinical data analysis. Furthermore, the proliferation, migration and invasion abilities of BC cells were assessed in vitro in the MDA-MB-231 and SK-BR3 BC cell lines. Luciferase reporter assay was carried out to unveil the association between DDX11-AS1, microRNA (miR)-30c-5p and metadherin (MTDH). Results DDX11-AS1 was significantly upregulated in BC tissues and cells. Additionally, bioinformatics analysis revealed that the expression levels of DDX11-AS1 were increased with enhanced pathological grading and lymph node metastasis. Furthermore, DDX11-AS1 knockdown markedly inhibited the proliferation, migration and invasion abilities of BC cells. Mechanistically, DDX11-AS1 could prevent the degradation of MTDH in BC via competitively binding with miR-30c-5p, which could act as a tumor promoter factor. Conclusion Collectively, the above results suggested that the DDX11-AS1/miR-30c-5p/MTDH axis could be associated with the progression of BC and DDX11-AS1 could be a potential biomarker and therapeutic target for BC. DDX11-AS1 miR-30c-5p metadherin breast cancer progression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Breast cancer (BC) is the most common malignant tumor in females worldwide. In 2023, the incidence of breast malignant tumor in women was overtaken by lung cancer, while BC ranks second in mortality rate among female malignant tumors, second only to lung cancer [ 1 ]. Due to its high heterogeneity in terms of morphology and genetics, BC is characterized by enhanced metastatic potential, easy relapse and high drug resistance. Although treatment approaches continue to improve, recurrence and metastasis still occur and several patients may develop the above with disease progression [ 2 ]. Previous studies have shown that non-coding RNAs play an important role in the development of BC, but the exact mechanism is unclear [ 3 , 4 ]. Long noncoding RNAs (lncRNAs), which have no protein coding potential, represent RNAs that are > 200 nucleotides in length [ 5 ]. It has been reported that lncRNAs are involved in various biological processes, such as mRNA degradation, gene imprinting, splicing regulation, chromatin remodeling and cell cycle regulation [ 6 ], particularly in cancer [ 7 ]. The competing endogenous RNAs (ceRNAs) hypothesis suggested that lncRNAs, acting as endogenous RNAs, could competitively promote mRNA expression via bonging to particular microRNAs (miRs) [ 8 ]. This is also known as the miRNA sponge effect [ 9 , 10 ]. A previous study demonstrated that the lncRNA BCRT1/miR-1303/polypyrimidine tract-binding protein 3 axis could affect the progression of BC [ 11 ]. In addition, ZEB1-AS1 inhibition induced cisplatin sensitivity in BC via promoting the miR-129-5p-dependent downregulation of zinc finger E‑box‑binding homeobox 1 [ 12 ]. The above findings indicated that ceRNAs could be possibly involved in the initiation, as well as the development of BC [ 11 ]. However, ceRNAs, which are significantly associated with the prognosis of BC, need to be further investigated. It has been reported that lncRNA DDX11-AS1, also known as cohesion regulator non-coding RNA, is involved in several types of cancer, including hepatocarcinoma, lung cancer, colorectal carcinoma, renal carcinoma and bladder cancer [ 13 – 15 ]. Another study also revealed that DDX11-AS1 was associated with the resistance of BC cells to chemotherapy [ 16 , 17 ]. Nevertheless, the detailed molecular mechanism underlying the effect of DDX11-AS1 on BC oncogenesis remains unknown. Therefore, in the present, the expression levels of DDX11-AS1 in BC were analyzed using public microarray. Furthermore, the effect of DDX11-AS1, acting as a ceRNA through the miR-30c-5p/metadherin (MTDH)axis, on BC carcinogenesis was also investigated. Previous clinical studies have found that elevated expression of MTDH is associated with poorer prognosis in BC patients, and cellular assays have revealed that MTDH can promote BC progression and paclitaxel resistance through activation of the NF-κB pathway. In our study, we found that overexpression of MTDH could activate the NF-κB pathway by enhancing p65 phosphorylation (p-p65) and caused nuclear translocation of p65 [ 18 , 19 ]. The results of the current study highlighted the role of DDX11-AS1 as a possible and promising therapeutic target for BC. Materials and methods Patients and specimens. BC tissues (n = 36) and matched adjacent normal breast tissues (> 5 cm away from tumor location) were obtained from 36 patients who underwent surgery at The Fourth Hospital of Hebei Medical University (Shijiazhuang, China) between September and December 2022. All recruited participants did not accept any therapy prior surgery. Informed consent was obtained from all patients. Tissue samples harvested in the current study were stored at -80˚C in a refrigerator. The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University. The clinicopathological characteristics of the patients are listed in Table S1 . Bioinformatics analysis. The Gene Expression Omnibus (GEO) datasets, GSE156229 and GSE156229 ( http://www.ncbi.nlm.nih.gov/geo/ ), and The Cancer Genome Atlas (TCGA; http://cancergenome.nih.gov/ ) database were used to analyze the differential expression of lncRNAs or miRNAs between BC and normal matched tissues. RNA extraction and reverse transcription-quantitative PCR (RT-qPCR). A TRIzol® reagent (Takara Bio, Inc.) was used to extract total RNA from cells and tissues, according with the manufacturer's instructions. RNA quality and concentration were measured using the A260/A280 ratio on a microplate reader (Thermo Fisher Scientific, Inc.). A total of 2µg RNA was reverse-transcribed into complementary DNA using the Transcriptor First Strand cDNA Synthesis Kit (Tiangen Biotech Co., Ltd.) under the following thermocycling conditions: 42˚C for 15 min and 95˚C for 3 min. qPCR was performed with the GoTaq® Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression levels were measured using the 2 −ΔΔCq method [ 20 ]. Human GAPDH and U6 served as internal reference genes to calculate the relative expression of lncRNAs and miRNAs, respectively. The primer sequences used are listed in Table S2. Subcellular fractionation. To determine the cellular localization of lncRNA DDX11-AS1, RNA from cytoplasmic and nuclear fractions was isolated and purified using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek Corp.), according to the manufacturer's instructions. RT was performed using the extracted RNAs and the expression levels of lncRNA DDX11-AS1 were detected using RT-qPCR. U6 and GAPDH were used as nuclear and cytoplasmic control genes, respectively. Cell culture. The human breast epithelial MCF10A cell line and the BC cell lines, BT549(ER:-, PR:-, HER2:-), MCF-7(ER:+, PR:+, HER2:-), MDA-MB-453(ER:-, PR:-, HER2:-), MDA-MB-231(ER:-, PR:-, HER2:-), and SK-BR3(ER:-, PR:-, HER2:+), were originally purchased from Procell Life Science & Technology Co., Ltd. MCF-7 and MDA-MB-231 cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.), while BT549 and MDA-MB-453 cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.). Both media were supplemented with 10% fetal bovine serum (FBS; Biological Industries Israel Beit-Haemek Ltd.) and antibiotics (Beijing Solarbio Science & Technology Co., Ltd.). The SK-BR3 cell line was maintained in McCoy's 5A medium containing 10% FBS (both from Procell Life Science & Technology Co., Ltd.), while MCF10A cells were cultured in a special medium (Procell Life Science & Technology Co., Ltd.). All cell lines were cultured at 37˚C in an incubator with 5% CO 2 . Cell transfection. The small interfering RNA targeting DDX11-AS (si-DDX11-AS) and the pcDNA-DDX11-AS1 overexpression plasmid were purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd. The corresponding empty pcDNA vector and siRNA were used as negative controls (NC). The miR-30c-5p inhibitor, miR-NC inhibitor, miR-30c-5p mimics, miR-NC mimics and the MTDH overexpression and knockdown plasmids and their corresponding NCs were also synthesized by Shanghai Jikai Gene Chemical Technology Co., Ltd. The indicated sequences are listed in Table S3. MDA-MB-231 or SK-BR3 cells were transfected with the above plasmids using the FuGENE® HD Transfection Reagent (Promega Corporation), according to the manufacturer's protocol. The transfection efficiency was measured by RT-qPCR. Following transfection for 48–72 h, subsequent experiments were carried out. Cell Counting Kit 8 (CCK-8) assay. BC cells were seeded into 96-well plates at a density of ~ 2x10 3 cells/well and transfected for 24 h. Each group of cells was set up with six compound pores and tested. Cell proliferation was assessed using a CCK-8 assay, according to manufacturer's instructions. Briefly, the following experimental steps were repeated at 0, 24, 48, 72 and 96 h. Each well was supplemented with 10 µl CCK-8 reagent (Wuhan Boster Biological Technology, Ltd.) and the plates were then incubated for 2 h in a humified incubator with 5% CO 2 at 37˚C. The absorbance at a wavelength of 450 nm was measured in each well using the Tecan Infinite F50 microplate reader (Tecan Group, Ltd.). Colony formation assay. Transfected cells (2×10 3 cells/well) were inoculated into 6-well plates and incubated with 10% FBS for 14 days. Then, colonies were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet dye (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min. Colonies consisted of ≥ 50 cells were counted and analyzed using Image J software (NIH). Transwell invasion and migration assays. A Transwell chamber (Corning; Corning, Inc.) was coated with 20 µl Matrigel (1 mg/ml; Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's instructions. For migration (without Matrigel) and invasion (with Matrigel; MilliporeSigma) assays, 4x10 4 BC cells were seeded into the upper chamber of the Transwell insert in 200 µl FBS-free medium. The lower chamber was supplemented with 600 µl 10% FBS medium as a chemoattractant. Following incubation at 37˚C for the indicated time points, non-invading cells were removed using a cotton swab. Cells adhering to the bottom surface of the membrane were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet for 20 min. The stained cells were counted using ImageJ software (NIH). Wound healing assay. Transfected cells were cultured in 6-well plates until reached 100% confluency. Subsequently, the cell monolayer was scraped linearly using a 10-µl pipette tip prior rinsing away any isolated cells with PBS. Subsequently, the remaining cells were cultured in FBS-free medium and then the artificial wound closure was observed. Images of the migrated cells were captured at 0, 24 or 48 h. The width of the wound area was measured by ImageJ software (NIH) and the mobility ratio was determined using the following formula: Mobility=(scratch width at 0 h-scratch width at 24 or 48 h)/scratch width at 0 h x100%. Luciferase reporter assay. The wild type (WT) and mutant (MUT) lncRNA DDX11-AS1 sequences and the 3'-untranslated regions (3'-UTR) of WT or MUT MTDH encompassing miR-30c-5p binding sites were inserted into the GP-miRGLO luciferase vector (Shanghai GenePharma Co., Ltd.). HEK293T cells were co-transfected with miR-30c-5p or miR-NC mimics and luciferase reporter plasmids for 48 h at 37˚C using the FuGENE® HD Transfection Reagent (Promega Corporation). Cells were harvested and the luciferase activity was measured using a Dual-luciferase reporter gene assay kit (Shanghai GenePharma Co., Ltd.), according to the manufacturer's instructions. Western blot assay. Cell lysis and protein separation was carried out using a RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with protease and phosphatase inhibitor intact tablets (Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration was measured using a BCA Protein Assay kit (Wuhan Boster Biological Technology, Ltd.). Total proteins were separated by 10% SDS-PAGE and were then electrotransfered onto PVDF membranes. Following blocking with 5% skimmed milk powder for 2 h at room temperature, the PVDF membranes were then incubated with primary antibodies against MTDH (dilution, 1:1000; rabbit; cat. no. 13860-1-AP), IκBα (dilution, 1:1000; rabbit; cat. no. 10268-1-AP), phosphorylation of IκBα (p-IκBα) (dilution, 1:5000; rabbit; cat. no. 82349-1-RR), p65 (dilution, 1:2000; rabbit; cat. no.10745-1-AP), p-p65 (dilution, 1:2000; rabbit; cat. no.82335-1-RR), Lamin B1 (dilution, 1:1000; rabbit; cat. no.12987-1-AP), and GAPDH (dilution, 1:10000; rabbit; cat. no.10494-1-AP; all from Proteintech Group, Inc.) at 4˚C overnight. The following day, the membranes were washed thrice with TBS-Tween for 10 min and were then incubated with the corresponding secondary antibodies at room temperature for 2 h. Finally, the protein bands were visualized using the Chemidoc XRS imaging system (Bio-Rad Laboratories) with an ECL solution. Statistical analysis. All statistical analyses were performed using the SPSS 26.0 (IBM Corp.) and GraphPad Prism 8 (GraphPad Software, Inc.) software. All measured data are expressed as the mean ± standard deviation (SD). The differences among multiple groups were compared by one-way ANOVA and Tukey's post hoc test or χ 2 test. Pearson correlation analysis was carried out to assess the association between DDX11-AS1 and miR-30c-5p in cell lines. All experiments were performed in at least three independent replicates. P < 0.05 was considered to indicate a statistically significant difference. Results DDX11-AS1 is upregulated in BC . To predict the key lncRNAs, which could be involved in BC progression, the public microarray GEO dataset GSE156229 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE156229 ), containing the differential lncRNA expression profiles between tumor and normal tissues from patients with BC were analyzed (fold change, ≥ 2 and P < 0.05; Fig. 1 a). The analysis predicted that lncRNA DDX11-AS1 was significantly differentially expressed in BC tissues. RNA-seq data from a BC cohort in the TCGA database also revealed that DDX11-AS1 expression was higher in BC tissues compared with normal breast tissues, thus suggesting that DDX11-AS1 could promote the progression of BC (P < 0.001; Fig. 1 b). In addition, RT-qPCR analysis showed that DDX11-AS1 was notably upregulated in BC tissues derived from 36 patients with BC compared with normal tissues (P < 0.001; Fig. 1 d). The clinicopathological characteristics of patients with BC are listed in Table 1. Furthermore, higher DDX11-AS1 expression levels were associated with advanced-stage (III) and lymph node-metastasis in patients with BC (P < 0.001; Fig. 1 e and f). Simultaneously, compared with MCF10A normal breast epithelial cells, DDX11-AS1 expression levels were significantly enhanced in the BT549, MDA-MB-231, MCF7, MDA-MB-453 and SK-BR3 BC cell lines. The results showed the highest expression in SK-BR3 cell line and slightly lower expression in BT549 and MDA-MB-231, but the difference was not significant. In addition, we verified that both BT549 and MDA-MB-231 functional results were statistically significant by pre-experiment, but the difference of MDA-MB-231 was more significant, so we selected these two cell lines for the next validation (P < 0.05; Fig. 1 c). Collectively, the above findings indicated that the DDX11-AS1 expression levels were increased in BC tissues and BC cell lines, and it could play a potential carcinogenic role in BC progression. DDX11-AS1 promotes BC cell proliferation, colony formation, migration and invasion. To evaluate the particular effects of DDX11-AS1 on BC, MDA-MB-231 cells were transfected with the pcDNA3.1 overexpression plasmid encompassing the DDX11-AS1 sequence. In addition, SK-BR3 cells were transfected with siRNA clones targeting DDX11-AS1 (si-DDX11-AS1-1,2,3). The above two cell lines were the most efficiently transfected ones. RT-qPCR analysis showed that DDX11-AS1 was successfully upregulated in MDA-MB-231 cells and downregulated in SK-BR3 cells (P < 0.05; Fig. 2 a and c). Since si-DDX11-AS1-2 exhibited the most potent silencing activity, this siRNA clone was used for the subsequent functional experiments. Therefore, CCK-8 assays demonstrated that SK-BR3 cell transfection with si-DDX11-AS1-2 significantly inhibited cell proliferation, while DDX11-AS1 overexpression in MDA-MB-231 cells showed the opposite effect (P < 0.05; Fig. 2 b and d). Consistently, clone formation assays further verified that DDX11-AS1 knockdown significantly inhibited SK-BR3 BC cell proliferation, while DDX11-AS1 overexpression significantly promoted the colony formation ability of MDA-MB-231 cells (P < 0.001; Fig. 2 e-f). Furthermore, wound healing and Transwell assays were performed to assess the effects of DDX11-AS1 on the invasion and migration abilities of BC cells. The results revealed that DDX11-AS1 overexpression in MDA-MB-231 cells promoted cell invasion and migration, while SK-BR3 cell transfection with si-DDX11-AS1-2 yielded the opposite results (P < 0.001; Fig. 2 g-j). DDX11-AS1 sponges miR-30c-5p in BC cells . It is widely accepted that the functions and mechanisms of lncRNAs are associated with their particular cellular localization [ 21 ]. Therefore, a subcellular distribution assay was performed to verify the subcellular localization of DDX11-AS1 in BC cells. DDX11-AS1 could be detected in both the nuclear and cytoplasmatic fractions of MDA-MB-231 and SK-BR3 cells (P < 0.05; Fig. 3 a and b). It was therefore hypothesized that DDX11-AS1 could act as a ceRNA to sponge miRNAs. To further verify the mechanism underlying the effect of DDX11-AS1 on BC, the expression profile of miRNAs was analyzed using the GEO GSE156229 database (Fold change, ≤-2 and P < 0.05; Fig. 3 c). Additionally, the StarBase V3.0 database predicted that DDX11-AS1 encompassed binding sites for both miR-30c-5p and miR-30e-5p. In addition, bioinformatics analysis revealed that miR-30c-5p was significantly downregulated in the GEO GSE156229 dataset. However, the expression levels of miR-30e-5p were higher (P < 0.05; Fig. 3 d and e). Additionally, the expression levels of miR-30c-5p were decreased in BC tissues compared with normal tissues. The above finding was consistent with that observed in the GEO dataset (P < 0.001; Fig. 3 f). To verify the results provided by the bioinformatics analysis, DDX11-AS1 was silenced in SK-BR3 cells and overexpressed in MDA-MB-231 cells. RT-qPCR analysis showed that miR-30c-5p was upregulated in DDX11-AS1-depleted BC cells and downregulated in DDX11-AS1 overexpressing BC cells (P < 0.05; Fig. 3 g-h). The above finding verified that miR-30c-5p could be a potential target of DDX11-AS1 (Fig. 3 i). Subsequently, dual-luciferase reporter gene assays were performed in 293T cells to confirm whether DDX11-AS1 could sponge miR-30c-5p. Therefore, the luciferase activity of the DDX11-AS1-WT reporter plasmid was significantly reduced in cells co-transfected with miR-30c-5p mimics, while this inhibition was eliminated when the binding site of miR-30c-5p in DDX11-AS1 sequence was mutated (P < 0.001; Fig. 3 j). The aforementioned results verified that DDX11-AS1 could sponge miR-30c-5p to inhibit its expression. miR-30c-5p silencing abolishes the suppressive effect of DDX11-AS1 knockdown on BC cells. Subsequently, rescue experiments were carried out to further investigate whether DDX11-AS1 could promote the progression of BC via regulating miR-30c-5p. CCK-8 and colony formation assays demonstrated that miR-30c-5p silencing partially reversed the inhibitory effect of DDX11-AS1-2 silencing on cell proliferation, while miR-30c-5p overexpression could rescue the effect of DDX11-AS1 overexpression (P < 0.01; Fig. 4 a-f). Additionally, the wound healing assays displayed the same results (P < 0.001; Fig. 4 g-h). In general, the above findings suggested that miR-30c-5p could be an inhibitory target of DDX11-AS1 in BC. DDX11-AS1 sponges miR-30c-5p to regulate the expression of MTDH in BC cells . Bioinformatics analysis using the miRDB, miRWalk, miRanda and TargetScan databases predicted that MTDH could be a potential target of miR-30c-5p (Fig. 5 a). Furthermore, the analysis also revealed that MTDH expression was positively and negatively associated with that of DDX11-AS1 and miR-30c-5p, respectively (P < 0.01; Fig. 5 c and d). In addition, the TargetScan database predicted that MTDH encompassed a sequence complementary to miR-30c-5p. This result suggested that MTDH could act as a potential target of miR-30c-5p and it was therefore selected for further investigation. Therefore, luciferase assays showed that miR-30c-5p overexpression reduced the luciferase activity of the WT MTDH reporter plasmid, but not that of the MUT reporter plasmid, thus further verifying that MTDH was a direct target of miR-30c-5p (P < 0.001; Fig. 5 b and e). The effect of DDX11-AS1 on the protein expression levels of MTDH was then assessed by western blot analysis. The results demonstrated that the protein expression levels of MTDH were significantly enhanced by DDX11-AS1 overexpression and miR-30c-5p knockdown, while they were reduced by DDX11-AS1 knockdown and miR-30c-5p overexpression.(P < 0.05; Fig. 5 f-i). DDX11-AS1/miR-30c-5p/MTDH axis promotes the progression of BC via regulating the activation of the NF-κB signaling pathway . To determine the role of the DDX11-AS1/miR-30c-5p/MTDH axis in BC, MDA-MB-231 cells were co-transfected with miR-30c-5p mimics with or without a MTDH overexpression plasmid. In addition, SK-BR3 cells were transfected with miR-30c-5p inhibitor with or without a siRNA clone targeting MTDH. The transfection efficiency was determined by RT-qPCR (P < 0.001; Fig. 6 a and c). The results showed that miR-30c-5p overexpression inhibited BC cell proliferation and this effect was reversed by MTDH overexpression. On the contrary, transfection of SK-BR3 cells with miR-30c-5p inhibitors significantly promoted cell proliferation, which was relieved by MTDH knockdown (P < 0.01; Fig. 6 b and d, e and f). Furthermore, miR-30c-5p overexpression attenuated BC cell migration and invasion, while miR-30c-5p knockdown enhanced the above processes. These effects were abolished by MTDH overexpression or knockdown, respectively (P < 0.001; Fig. 6 g-j). In addition, western blot analysis demonstrated that overexpression of MTDH promoted ikba degradation, enhanced p-p65 to activate the NF-κB pathway, and caused nuclear translocation of p65, while knockdown of MTDH had the opposite result. (P < 0.01; Fig. 6 k and l). The above data suggested that DDX11-AS1 exerted a cancer promotive effect on BC via downregulating MTDH through competitively binding to miR-30c-5p in vitro . Discussion lncRNA, as a non-coding RNA, was involved in a range of biological functions related to carcinogenesis and had therefore received increasing attention [ 22 , 23 ]. Accumulated evidence has indicated that lncRNAs located in the cytoplasm serve as ceRNAs to protect target mRNAs from repression, while they are involved in gene regulation at the post-transcriptional level [ 8 , 17 ]. The significant role of lncRNAs as ceRNAs has been previously verified in BC. Therefore, a previous study demonstrated that the hypoxia inducible factor-1/lncRNA BCRT1/miR-1303/polypyrimidine tract-binding protein 3 axis could further accelerate the progression of BC via promoting the exosome-mediated M2 macrophage polarization [ 11 ]. Additionally, the lncRNA-MAFG-AS1/miR-339-5p/cyclin-dependent kinase 2 axis could promote the progression of ER + BC and the resistance of BC cells to tamoxifen [ 24 ]. Another study revealed that lncRNA-NRON inhibited BC development via regulating the miR-302b/serine- and arginine-rich splicing factor 2 axis [ 25 ]. Other studies also demonstrated that DDX11-AS1 was upregulated in drug resistant BC cell lines, thus suggesting that it could be involved in chemotherapy resistance in BC [ 16 , 17 ]. Nonetheless, the biological significance of DDX11-AS1 in BC remains largely unknown. To the best of our knowledge, the current study was the first to show that DDX11-AS1 was significantly upregulated in BC tissues. At the same time, the DDX11-AS1 expression levels were increased with higher pathological stage and lymph node metastasis. Functional studies demonstrated that DDX11-AS1 overexpression facilitated the proliferation, invasion and migration of BC cells in vitro , thus supporting the tumor-promotive role of DDX11-AS1 in BC. Previous studies also indicated that uncontrolled NF-κB expression could be associated with the development of BC [ 26 , 27 ]. Therefore, the activation of NF-κB could inhibit apoptosis and promote metastasis, thereby providing BC with a more aggressive phenotype [ 28 ]. The present study revealed that DDX11-AS1 could promote BC progression via regulating the NF-κB signaling pathway. Herein, subcellular distribution assays also showed that DDX11-AS1 was expressed in the cytoplasm of BC cells, thus suggesting that it could act as a ceRNA to sponge miRNAs. Previous studies demonstrated that miR-30c-5p could inhibit the progression of different types of cancer, such as oral squamous cell carcinoma, gastric cancer and clear cell renal cell carcinoma [ 29 – 31 ]. Pei et al [ 32 ] indicated that miR-30c-5p could restrain tumor metastasis and BC cell migration via targeting coactosin-like protein 1. Furthermore, Yen et al [ 33 ] demonstrated that isolinderalactone inhibited the STAT3 signaling pathway via regulating suppressor of cytokine signaling 3 and miR-30c in BC. Herein, bioinformatic analysis using the StarBase V3.0 database predicted that miR-30c-5p encompassed a complementary sequence of DDX11-AS1. This finding was further verified by dual-luciferase reporter assays. Additionally, the results showed that DDX11-AS1 was upregulated and miR-30c-5p was downregulated in BC tissues, further supporting that DDX11-AS1 expression was negatively associated with that of miR-30c-5p. Therefore, miR-30c-5p overexpression could partially reverse the promotive effect of DDX11-AS1 on the behavior of BC cells. Overall, the results suggested that DDX11-AS1 could sponge miR-30c-5p to inhibit its expression. MTDH is ubiquitously expressed in almost all human tissues at variable levels, while its expression levels are dramatically enhanced in cancer [ 34 ]. A study revealed that the abundance of MTDH was positively associated with advanced clinical stages, clinicopathological features, distant metastasis and poor survival in patients with BC [ 35 ]. MTDH expression in cancer cells can be modulated through a miRNA-mediated post-translational process [ 36 ]. For example, miR-375 could attenuate the growth of hepatocellular carcinoma via downregulating MTDH [ 37 ]. In the present study, dual-luciferase reporter assays combined with bioinformatics analysis indicated that MTDH could be a direct target of miR-30c-5p. Besides, DDX11-AS1 positively regulated MTDH expression in BC. Furthermore, the effects of the DDX11-AS1/miR-30c-5p/MTDH axis in the progress of BC were verified by in vitro rescue experiments, and our results revealed that overexpression of MTDH could activate the NF-κB pathway by enhancing p-p65 and caused nuclear translocation of p65, which is consistent with previous studies [ 38 ]. Previous studies showed that MTDH and NF-κB were involved in paclitaxel and doxorubicin drug resistance, and MTDH overexpression may mediate trastuzumab resistance by increasing p65 nuclear translocation [ 38 – 40 , 18 ]. Additionally, DDX11-AS1 was upregulated in drug resistant BC cell lines, thus suggesting that DDX11-AS1 could be involved in the resistance of BC cells to paclitaxel, doxorubicin and trastuzumab through the MTDH/NF-κB axis [ 16 , 17 , 38 ]. It was therefore hypothesized that the DDX11-AS1/miR-30c-5p/MTDH axis could serve a key role in the chemoresistance of BC cells. However, our study did not have the means to validate the correlation between DDX11-AS1 and the prognosis of BC patients due to the limitation of available specimens.. In conclusion, the present study demonstrated that DDX11-AS1 acted as a tumor promoter factor in BC. As a sponge of miR-30c-5p, DDX11-AS1 weakened its inhibitory effect on MTDH and promoted cell proliferation, migration, invasion and the activation of the NF-κB signaling pathway in BC. Overall, the present study suggested that DDX11-AS1 could be a potential target in BC therapy. Declarations Acknowledgements Not applicable. Funding This study was supported by the Provincial Health Commission of Hebei province (grant no. 20230485), the Natural Science Foundation of Hebei Province (grant nos. H2020206365 and H2021206071), the S&T Program of Hebei (grant no. 236Z7719G), the Special Fund for Clinical Research of the Wu Jieping Medical Foundation (grant no. 320.6750.2020/07/17) and the Bethune Cancer Basic Research Program (grant no. BCF-NH-ZL-20201119-013). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors' contributions ZS conceived, designed and oversaw the study. SL collected the clinical specimens and data. YTL and MSZ conducted the experiments. YTL and MSZ analyzed and interpreted the data. YTL wrote the manuscript. ZS revised the manuscript critically for significant intellectual content. YTL and MSZ confirmed the authenticity of all raw data. All authors read and approved the final manuscript. Ethics approval and consent to participate All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of 2021KY056. Patient consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Siegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023. Ca-Cancer J Clin 73(1): 17-48. Saad ED, Katz A, Buyse M (2010) Overall survival and post-progression survival in advanced breast cancer: a review of recent randomized clinical trials. J Clin Oncol 28(11): 1958-62. Liu P, Wang ZH, Ou XQ, Wu P, Zhang Y, Wu S, et al(2022) The FUS/circEZH2/KLF5/ feedback loop contributes to CXCR4-induced liver metastasis of breast cancer by enhancing epithelial-mesenchymal transition. Mol Cancer 21(1): 198. Zeng Y, Du W, Huang ZY, Wu S, Ou XQ, Zhang JH, et al(2023) Hsa_circ_0060467 promotes breast cancer liver metastasis by complexing with eIF4A3 and sponging miR-1205. Cell Death Discov 9(1): 153. Chen Z, Chen X, Chen P, Yu S, Nie F, Lu B, et al (2017) Long non-coding RNA SNHG20 promotes non-small cell lung cancer cell proliferation and migration by epigenetically silencing of P21 expression. Cell Death Dis 8(10): e3092. Li D, Cheng M, Niu Y, Chi X, Liu X, Fan J, et al (2017) Identification of a novel human long non-coding RNA that regulates hepatic lipid metabolism by inhibiting SREBP-1c. Int J Biol Sci 13(3): 349-357. Prensner JR, Chinnaiyan AM (2011). The emergence of lncRNAs in cancer biology. Cancer Discov 1(5): 391-407. Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP (2011) A ceRNA hypothesis: the rosetta stone of a hidden RNA language?. Cell 146(3): 353–358. Paraskevopoulou MD, Hatzigeorgiou AG (2016) Analyzing MiRNA-LncRNA Interactions. Methods Mol Biol 1402: 271–286. An Y, Furber KL, Ji S (2017) Pseudogenes regulate parental gene expression via ceRNA network. J Cell Mol Med 21(1): 185–192. Liang Y, Song X, Li Y, Chen B, Zhao W, Wang L, et al (2020) LncRNA BCRT1 promotes breast cancer progression by targeting miR-1303/PTBP3 axis. Mol Cancer 19(1): 85. Gao J, Yuan Y, Zhang L, Yu S, Lu J, Feng J, et al (2020) Inhibition of ZEB1-AS1 confers cisplatin sensitivity in breast cancer by promoting microRNA-129-5p-dependent ZEB1 downregulation. Cancer Cell Int 20: 90. Wan T, Zheng J, Yao R, Yang S, Zheng W, Zhou P (2021) LncRNA DDX11-AS1 accelerates hepatocellular carcinoma progression via the miR-195-5p/MACC1 pathway. Ann Hepatol 20: 100258. Liu J, Yang X, Gao S, Wen M, Yu Qiong (2023) DDX11-AS1 modulates DNA damage repair to enhance paclitaxel resistance of lung adenocarcinoma cells. Pharmacogenomics 24(3): 163-172. Marchese FP, Grossi E, Marín-Béjar O, Bharti SK, Raimondi I, González J, et al (2016) A long noncoding RNA regulates sister chromatid cohesion. Mol Cell 63(3): 397–407. Si X, Zhang G, Li M, Yao M, Shi X, Dong Z, et al (2023) DDX11-AS1 Promotes Chemoresistance through LIN28A-Mediated ATG12 mRNA Stabilization in Breast Cancer. PHARMACOLOGY 108(1): 61-73. Liang M, Zhu B, Wang M, Jin J (2022) Knockdown of long non‑coding RNA DDX11‑AS1 inhibits the proliferation, migration and paclitaxel resistance of breast cancer cells by upregulating microRNA‑497 expression. Mol Med Rep 25(4): 123. Yang L, Tian Y, Leong WS, Song H, Yang W, Wang M, et al (2018) Efficient and tumor-specific knockdown of MTDH gene attenuates paclitaxel resistance of breast cancer cells both in vivo and in vitro. Breast Cancer Res 20(1): 113. Li J, Zhang N, Song LB, Liao WT, Jiang LL, Gong LY, et al (2008) Astrocyte elevated gene-1 is a novel prognostic marker for breast cancer progression and overall patient survival. Clin Cancer Res 14(11): 3319-26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25(4): 402–408. Zhang K, Shi Z, Chang Y, Hu Z, Qi H, Hong W (2014) The ways of action of long non-coding RNAs in cytoplasm and nucleus. Gene 547(1): 1-9. Sun M, Nie F, Wang Z, De W (2016) Involvement of lncRNA dysregulation in gastric cancer. Histol And Histopathol 31(1): 33–39. Cen SZ, Peng XJ, Deng JW, Jin HY, Deng ZN, Lin XH, et al (2023) The role of AFAP1-AS1 in mitotic catastrophe and metastasis of triple-negative breast cancer cells by activating the PLK1 signaling pathway. Oncol Res 31(3): 375-388. Feng J, Wen T, L Z, Feng L, Zhou L, Yang Z, et al (2020) Cross-talk between the ER pathway and the lncRNA MAFG-AS1/miR-339-5p/ CDK2 axis promotes progression of ER+ breast cancer and confers tamoxifen resistance. Aging (Albany NY) 12(20): 20658-20683. Mao Q, Li L, Zhang C, Sun Y, Liu S, Li Y, et al (2020) Long non coding RNA NRON inhibited breast cancer development through regulating miR-302b/SRSF2 axis. Am J Transl Res 12(8): 4683-4692. Romieu-Mourez R, Kim DW, Shin SM, Demicco EG, Landesman-Bollag E, Seldin DC, et al (2003) Mouse mammary tumor virus c-rel transgenic mice develop mammary tumors. Mol Med Rep 23(16): 5738-5754. Zhou J, Hao Z, Gu P , Bai J, Margolick JB, Zhang Y (2008) NF-kappaB pathway inhibitors preferentially inhibit breast cancer stem-like cells. Breast Cancer Res Tr 111(3): 419-427. Wu JT, Kral JG (2005) The NF-kappaB/IkappaB signaling system: a molecular target in breast cancer therapy. J Surg Res 123(1): 158-169. Mehterov N, Vladimirov B, Sacconi A, Pulito C, Rucinski M, Blandino G, et al (2021) Salivary miR-30c-5p as Potential Biomarker for Detection of Oral Squamous Cell Carcinoma. Biomedicines 9(9): 1079. Cao J, Li G, Han M, Xu H, Huang K (2017) MiR-30c-5p suppresses migration, invasion and epithelial to mesenchymal transition of gastric cancer via targeting MTA1. Biomed Pharmacother 93: 554-560. Outeiro-Pinho G, Barros-Silva D, Moreira-Silva F, Lobo J, Carneiro I, Morais A, et al (2022) Epigenetically-regulated miR-30a/c-5p directly target TWF1 and hamper ccRCC cell aggressiveness. Transl Res 249: 110-127. Pei B, Li T, Qian Q, Fan W, He X, Zhu Y, et al (2020) Downregulation of microRNA-30c-5p was responsible for cell migration and tumor metastasis via COTL1-mediated microfilament arrangement in breast cancer. Gland Surg 9(3): 747-758. Yen MC, Shih YC, Hsu YL, Lin ES, Lin YS, Tsai EM, et al (2016) Isolinderalactone enhances the inhibition of SOCS3 on STAT3 activity by decreasing miR-30c in breast cancer. Oncol Rep 35(3): 1356-64. Kang DC, Su ZZ, Sarkar D, Emdad L, Volsky DJ, Fisher PB (2005) Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1. Gene 353(1): 8–15. Wan L, Kang Y (2013) Pleiotropic roles of AEG-1/MTDH/LYRIC in breast cancer. Adv Cancer Res 120: 113-134. Khan M, Sarkar D (2021) The scope of astrocyte elevated Gene-1/Metad-herin (AEG-1/MTDH) in Cancer Clinicopathology: A Review. Genes (Basel) 12(2): 308. He X, Chang Y, Meng F, Wang M, Xie Q, Tang F, et al (2012) MicroRNA-375 targets AEG-1 in hepatocellular carcinoma and suppresses liver cancer cell growth in vitro and in vivo. Oncogene 31(28): 3357–3369. Du C, Yi X, Liu W, Han T, Liu Z, Ding Z, et al (2014) MTDH mediates trastuzumab resistance in HER2 positive breast cancer by decreasing PTEN expression through an NF-κB-dependent pathway. BMC Cancer 14: 869. Song Z, Wang Y, Li C, Zhang D, Wang X (2015) Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin. Plos One 10(5): e0127599. Abdin SM, Tolba MF, Zaher DM, Omar HA (2021) Nuclear factor-κB signaling inhibitors revert multidrug-resistance in breast cancer cells. Chem-biol Interact 340: 109450. Additional Declarations No competing interests reported. Supplementary Files supplementary.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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3822928","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":264471670,"identity":"d9847c2c-3270-4f2f-8803-839671eb3acb","order_by":0,"name":"Yanting Li","email":"","orcid":"","institution":"the Second Hospital of Hebei Medical University，Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Yanting","middleName":"","lastName":"Li","suffix":""},{"id":264471671,"identity":"da2f2c7d-a7ca-4a31-a4f7-1970deebdced","order_by":1,"name":"Mengsi Zhou","email":"","orcid":"","institution":"the Second Hospital of Hebei Medical University，Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Mengsi","middleName":"","lastName":"Zhou","suffix":""},{"id":264471672,"identity":"ea5a2424-8c55-49b8-a0c1-8be3e6941399","order_by":2,"name":"Liu Yang","email":"","orcid":"","institution":"Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Yang","suffix":""},{"id":264471673,"identity":"b83fbae0-5939-4fdd-8d26-3c727dde4b66","order_by":3,"name":"Shuo Liu","email":"","orcid":"","institution":"Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Liu","suffix":""},{"id":264471674,"identity":"921b253d-5d4c-4773-a7c3-7be8b1c14d3f","order_by":4,"name":"Lixian Yang","email":"","orcid":"","institution":"Xingtai People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lixian","middleName":"","lastName":"Yang","suffix":""},{"id":264471675,"identity":"76155591-aba4-4286-aff7-fa82774767ae","order_by":5,"name":"Bin Xu","email":"","orcid":"","institution":"Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Xu","suffix":""},{"id":264471676,"identity":"6611881c-ecfa-4001-8e8b-2e11f54a523d","order_by":6,"name":"Xiaolong Li","email":"","orcid":"","institution":"the Fourth Hospital of Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Xiaolong","middleName":"","lastName":"Li","suffix":""},{"id":264471677,"identity":"9261d76c-a6f4-429a-9372-e50bb6db1729","order_by":7,"name":"Haijun Zhao","email":"","orcid":"","institution":"the Fourth Hospital of Shijiazhuang","correspondingAuthor":false,"prefix":"","firstName":"Haijun","middleName":"","lastName":"Zhao","suffix":""},{"id":264471678,"identity":"03e06f5e-f50b-4542-bc00-2240c91156f6","order_by":8,"name":"Zhenchuan Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYLACxgaGBH4JMFNChngtkjNAFIMED/FaDG6AtTAQ1iLf3nv45c8ddnnGt5uPP7pRY8HDwH746AZ8WgzOnEuzkDyTXGx251hic84xoMN40tJu4NUikWNmYNh2IHHbjRzD5hw2oBYJHjO8WuRnALUkArVsngHS8o8ILQw3cowfHARq2SAB1JLbRoQWgzNnzBgb25ITZ9xIS5yd2yfBw0bIL/LtPcYff7bZJfbPSD7wOedbnRw/++Fj+B3GwMAmgcoloBwEmD8QoWgUjIJRMApGMgAAlLFL3tkt4lMAAAAASUVORK5CYII=","orcid":"","institution":"the Fourth Hospital of Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhenchuan","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2023-12-30 02:29:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3822928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3822928/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49137267,"identity":"bb661d61-9416-4ca8-8117-d2f0b0b79a53","added_by":"auto","created_at":"2024-01-03 17:27:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":400491,"visible":true,"origin":"","legend":"\u003cp\u003eDDX11-AS1 expression was higher in BC tissues and cell lines. (a) The differential lncRNAs with fold change ≥2 and p\u0026lt;0.05 between BC samples and normal samples in public GSE156229 dataset. (b) The expression of DDX11-AS1 in BC and normal tissues in TCGA database. (c) DDX11-AS1 expression analysis in five BC cell lines and normal MCF-10A cells. (d) RT-PCR analysis was used to detect the expression of DDX11-AS1 in tumour tissues and normal tissues in 36 BC patients. (e) Differential expression of DDX11-AS1 with different pathological grades in BC tissues. (f) Differential expression of DDX11-AS1 with or without lymph node metastasis in BC tissues. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3822928/v1/05e891f3b70d73d76340ece7.png"},{"id":49137704,"identity":"6e8512e0-3d2c-4cb8-9f2c-01d3a4100445","added_by":"auto","created_at":"2024-01-03 17:35:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2190082,"visible":true,"origin":"","legend":"\u003cp\u003eDDX11-AS1 knockdown inhibited BC cell proliferation in vitro.Si-DDX11-AS1 or si-NC was transfected into SK-BR3 cells,and DDX11-AS1 or pcDNA3.1 was introduced into MDA-MB-231 cells.(a) Transfection efficiency of DDX11-AS1 into MDA-MB-231 cells by qRT-PCR. (b) Cell proliferation by CCK-8 assay in MDA-MB-231 cells after transfection. (c) Transfection efficiency of si-DDX11-AS1 into SK-BR3 cells. (d) Cell proliferation by CCK-8 assay in SK-BR3 cells after transfection. (e-f) Cell proliferation by colony formation assay in MDA-MB-231 cells and SK-BR3 cells. (g-h)Cell migration capacities were conducted by wound healing assays after transfection in MDA-MB-231 cells and SK-BR3 cells. Scale bar, 200µm. (i-j) Cell invasion and migration abilities were indicated by transwell assays in each group. Scale bar, 200 µm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3822928/v1/62610df8a85a10ac17c45096.png"},{"id":49137265,"identity":"a49ef70b-e090-4d7f-883e-d778e714417f","added_by":"auto","created_at":"2024-01-03 17:27:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":599539,"visible":true,"origin":"","legend":"\u003cp\u003eDDX11-AS1 interacted with miR-30c-5p to inhibit its expression. (a-b) The expression level of DDX11-AS1 in the subcellular fractions of MDA-MB-231 and SK-BR3 cells was detected by qRT-PCR. U6 and GAPDH were used as nuclear and cytoplasmic markers, respectively. (c) The diferential miRNAs with fold change ≤-2 and p\u0026lt;0.05 between BC samples and normal samples in public GSE156229 dataset. (d-e) The expression of miR-30c-5p and miR-30e-5p in BC and normal tissues in public GSE156229 dataset. (f) The expression of miR-30c-5p in tumour tissues and normal tissues in 15 BC patients by RT-qPCR. (g-h) The effect of si-DDX11-AS1 and OE-DDX11-AS1 on the expressions of miR-30c-5p in SK-BR3 and MDA-MB-231 cells. DDX11-AS1 and miR-30c-5p was negatively correlated. (i) Predicted binding sites on DDX11-AS1 to sponge miR-30c-5p. (j) Dual-luciferase reporter assays in HEK293T was carried out to investigate the interplay between miR-30c-5p and DDX11-AS1.*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3822928/v1/f7ea464a7b75ed33adf289ce.png"},{"id":49137271,"identity":"6bf79682-b597-44ab-b9e9-da67974da362","added_by":"auto","created_at":"2024-01-03 17:27:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1258722,"visible":true,"origin":"","legend":"\u003cp\u003eDDX11-AS1 sponges miR-30c-5p in BC. Si-DDX11-AS1 or si-NC and miR-30c-5p inhibitor or NC inhibitor was cotransfected into SK-BR3 cells,and DDX11-AS1 or pcDNA3.1 and miR-30c-5p mimic or NC mimic was introduced into MDA-MB-231 cells. (a-f)CCK-8 assay and colony formation assay for SK-BR3 cells and MDA-MB-231 cells after cotransfection. (g-h) Cell migration capacities were conducted by wound healing assays after cotransfection. Scale bar, 200µm. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. OE-lnc: Overexpression of DDX11-AS1, miR mimic: miR-30c-5p mimic, NC-inh: NC-inhibitor, si-lnc: si-DDX11-AS1, miR inh: miR-30c-5p inhibitor.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3822928/v1/8f88c3c5556a95e540fd0ffb.png"},{"id":49137705,"identity":"5a471efe-9d22-4e31-8881-287d49ff705e","added_by":"auto","created_at":"2024-01-03 17:35:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":646182,"visible":true,"origin":"","legend":"\u003cp\u003eMTDH is a direct target of miR-30c-5p (a) Venn diagram represents the putative target genes of miR-30c-5p identified by miRWalk, miRDB, TargetScan, and miRanda. (b) Predicted binding sites and potential mutation sequences of miR-30c-5p binding sites in MTDH. (c-d) The effect of si-DDX11-AS1 and miR-30c-5p inhibitor on the expressions of MTDH in SK-BR3 cells. (e) The luciferase activity of WT or MUT luciferase plasmids after transfection with miR-30c-5p mimics in HEK293T cells. (f-i) western blot assays were used to determine the MTDH expression level in MDA-MB-231 and SK-BR3 cells cotransfected with DDX11-AS1 and miR-30c-5p mimic or si-DDX11-AS1 and miR-30c-5p inhibitor. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. OE-lnc: Overexpression of DDX11-AS1, si-lnc: si-DDX11-AS1, miR mimic: miR-30c-5p mimic, NC-inh: NC-inhibitor, miR inh: miR-30c-5p inhibitor.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3822928/v1/d4e4e5b9922eecf00073633d.png"},{"id":49137268,"identity":"54cb6f4a-063f-4391-b185-e8355366e31d","added_by":"auto","created_at":"2024-01-03 17:27:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2237523,"visible":true,"origin":"","legend":"\u003cp\u003eThe DDX11-AS1/miR-30c-5p/MTDH axis promotes progression in BC cells. si-MTDH or si-NC and miR-30c-5p inhibitor or NC inhibitor was cotransfected into SK-BR3 cells,and MTDH or empty vector and miR-30c-5p mimic or NC mimic was introduced into MDA-MB-231 cells. (a)The expression levels of miR-30c-5p in MDA-MB-231 cells by qRT-PCR. (b) CCK-8 assay for MDA-MB-231 cells after cotransfection. (c)The expression levels of miR-30c-5p in SK-BR3 cells by qRT-PCR. (d) CCK-8 assay in SK-BR3 cells after cotransfection. (e-f) The effects of miR-30c-5p and MTDH cotransfected on cell proliferation were examined by colony formation assay. (g-h) Cell migration capacities were conducted by wound healing assays after cotransfection. Scale bar, 200µm. (i-j) Cell invasion and migration abilities were indicated by transwell assays in each group. Scale bar, 200µm. (k-l) Western blot analysis of the protein expression levels of p65, p-p65, IκBα, p-IκBα, N-p65, C-p65, and MTDH after transfection. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. miR mimic: miR-30c-5p mimic, NC-inh: NC-inhibitor, si-lnc: si-DDX11-AS1, miR inh: miR-30c-5p inhibitor. p-p65: p65 phosphorylation, p-IκBα: phosphorylation of IκBα, C-p65: cytoplasm-p65, N-p65: nucleus-p65.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3822928/v1/d6b97babd978137c861f917a.png"},{"id":49184421,"identity":"e08a3027-82d3-44a8-ae13-87278efb9565","added_by":"auto","created_at":"2024-01-04 16:09:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3943274,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3822928/v1/e5b2e3ca-ec1b-49db-9c92-80be0a0b8a28.pdf"},{"id":49137270,"identity":"3cf02d0d-0d41-483c-bfe4-dffa3502bb60","added_by":"auto","created_at":"2024-01-03 17:27:22","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":24146,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-3822928/v1/cd6f1199f0dd76ac3a5683e5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"LncRNA DDX11-AS1 promotes breast cancer progression via targeting the miR-30c-5p/MTDH axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer (BC) is the most common malignant tumor in females worldwide. In 2023, the incidence of breast malignant tumor in women was overtaken by lung cancer, while BC ranks second in mortality rate among female malignant tumors, second only to lung cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Due to its high heterogeneity in terms of morphology and genetics, BC is characterized by enhanced metastatic potential, easy relapse and high drug resistance. Although treatment approaches continue to improve, recurrence and metastasis still occur and several patients may develop the above with disease progression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previous studies have shown that non-coding RNAs play an important role in the development of BC, but the exact mechanism is unclear [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLong noncoding RNAs (lncRNAs), which have no protein coding potential, represent RNAs that are \u0026gt;\u0026thinsp;200 nucleotides in length [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It has been reported that lncRNAs are involved in various biological processes, such as mRNA degradation, gene imprinting, splicing regulation, chromatin remodeling and cell cycle regulation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], particularly in cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The competing endogenous RNAs (ceRNAs) hypothesis suggested that lncRNAs, acting as endogenous RNAs, could competitively promote mRNA expression via bonging to particular microRNAs (miRs) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This is also known as the miRNA sponge effect [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A previous study demonstrated that the lncRNA BCRT1/miR-1303/polypyrimidine tract-binding protein 3 axis could affect the progression of BC [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, ZEB1-AS1 inhibition induced cisplatin sensitivity in BC via promoting the miR-129-5p-dependent downregulation of zinc finger E‑box‑binding homeobox 1 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The above findings indicated that ceRNAs could be possibly involved in the initiation, as well as the development of BC [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, ceRNAs, which are significantly associated with the prognosis of BC, need to be further investigated.\u003c/p\u003e \u003cp\u003eIt has been reported that lncRNA DDX11-AS1, also known as cohesion regulator non-coding RNA, is involved in several types of cancer, including hepatocarcinoma, lung cancer, colorectal carcinoma, renal carcinoma and bladder cancer [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Another study also revealed that DDX11-AS1 was associated with the resistance of BC cells to chemotherapy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nevertheless, the detailed molecular mechanism underlying the effect of DDX11-AS1 on BC oncogenesis remains unknown. Therefore, in the present, the expression levels of DDX11-AS1 in BC were analyzed using public microarray. Furthermore, the effect of DDX11-AS1, acting as a ceRNA through the miR-30c-5p/metadherin (MTDH)axis, on BC carcinogenesis was also investigated. Previous clinical studies have found that elevated expression of MTDH is associated with poorer prognosis in BC patients, and cellular assays have revealed that MTDH can promote BC progression and paclitaxel resistance through activation of the NF-κB pathway. In our study, we found that overexpression of MTDH could activate the NF-κB pathway by enhancing p65 phosphorylation (p-p65) and caused nuclear translocation of p65 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The results of the current study highlighted the role of DDX11-AS1 as a possible and promising therapeutic target for BC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003ePatients and specimens.\u003c/em\u003e BC tissues (n\u0026thinsp;=\u0026thinsp;36) and matched adjacent normal breast tissues (\u0026gt;\u0026thinsp;5 cm away from tumor location) were obtained from 36 patients who underwent surgery at The Fourth Hospital of Hebei Medical University (Shijiazhuang, China) between September and December 2022. All recruited participants did not accept any therapy prior surgery. Informed consent was obtained from all patients. Tissue samples harvested in the current study were stored at -80˚C in a refrigerator. The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University. The clinicopathological characteristics of the patients are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBioinformatics analysis.\u003c/em\u003e The Gene Expression Omnibus (GEO) datasets, GSE156229 and GSE156229 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and The Cancer Genome Atlas (TCGA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cancergenome.nih.gov/\u003c/span\u003e\u003cspan address=\"http://cancergenome.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database were used to analyze the differential expression of lncRNAs or miRNAs between BC and normal matched tissues.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRNA extraction and reverse transcription-quantitative PCR (RT-qPCR).\u003c/em\u003e A TRIzol\u0026reg; reagent (Takara Bio, Inc.) was used to extract total RNA from cells and tissues, according with the manufacturer's instructions. RNA quality and concentration were measured using the A260/A280 ratio on a microplate reader (Thermo Fisher Scientific, Inc.). A total of 2\u0026micro;g RNA was reverse-transcribed into complementary DNA using the Transcriptor First Strand cDNA Synthesis Kit (Tiangen Biotech Co., Ltd.) under the following thermocycling conditions: 42˚C for 15 min and 95˚C for 3 min. qPCR was performed with the GoTaq\u0026reg; Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression levels were measured using the 2\u003csup\u003e\u0026minus;ΔΔCq\u003c/sup\u003e method [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Human GAPDH and U6 served as internal reference genes to calculate the relative expression of lncRNAs and miRNAs, respectively. The primer sequences used are listed in Table S2.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSubcellular fractionation.\u003c/em\u003e To determine the cellular localization of lncRNA DDX11-AS1, RNA from cytoplasmic and nuclear fractions was isolated and purified using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek Corp.), according to the manufacturer's instructions. RT was performed using the extracted RNAs and the expression levels of lncRNA DDX11-AS1 were detected using RT-qPCR. U6 and GAPDH were used as nuclear and cytoplasmic control genes, respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell culture.\u003c/em\u003e The human breast epithelial MCF10A cell line and the BC cell lines, BT549(ER:-, PR:-, HER2:-), MCF-7(ER:+, PR:+, HER2:-), MDA-MB-453(ER:-, PR:-, HER2:-), MDA-MB-231(ER:-, PR:-, HER2:-), and SK-BR3(ER:-, PR:-, HER2:+), were originally purchased from Procell Life Science \u0026amp; Technology Co., Ltd. MCF-7 and MDA-MB-231 cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.), while BT549 and MDA-MB-453 cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.). Both media were supplemented with 10% fetal bovine serum (FBS; Biological Industries Israel Beit-Haemek Ltd.) and antibiotics (Beijing Solarbio Science \u0026amp; Technology Co., Ltd.). The SK-BR3 cell line was maintained in McCoy's 5A medium containing 10% FBS (both from Procell Life Science \u0026amp; Technology Co., Ltd.), while MCF10A cells were cultured in a special medium (Procell Life Science \u0026amp; Technology Co., Ltd.). All cell lines were cultured at 37˚C in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell transfection.\u003c/em\u003e The small interfering RNA targeting DDX11-AS (si-DDX11-AS) and the pcDNA-DDX11-AS1 overexpression plasmid were purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd. The corresponding empty pcDNA vector and siRNA were used as negative controls (NC). The miR-30c-5p inhibitor, miR-NC inhibitor, miR-30c-5p mimics, miR-NC mimics and the MTDH overexpression and knockdown plasmids and their corresponding NCs were also synthesized by Shanghai Jikai Gene Chemical Technology Co., Ltd. The indicated sequences are listed in Table S3. MDA-MB-231 or SK-BR3 cells were transfected with the above plasmids using the FuGENE\u0026reg; HD Transfection Reagent (Promega Corporation), according to the manufacturer's protocol. The transfection efficiency was measured by RT-qPCR. Following transfection for 48\u0026ndash;72 h, subsequent experiments were carried out.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell Counting Kit 8 (CCK-8) assay.\u003c/em\u003e BC cells were seeded into 96-well plates at a density of ~\u0026thinsp;2x10\u003csup\u003e3\u003c/sup\u003e cells/well and transfected for 24 h. Each group of cells was set up with six compound pores and tested. Cell proliferation was assessed using a CCK-8 assay, according to manufacturer's instructions. Briefly, the following experimental steps were repeated at 0, 24, 48, 72 and 96 h. Each well was supplemented with 10 \u0026micro;l CCK-8 reagent (Wuhan Boster Biological Technology, Ltd.) and the plates were then incubated for 2 h in a humified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37˚C. The absorbance at a wavelength of 450 nm was measured in each well using the Tecan Infinite F50 microplate reader (Tecan Group, Ltd.).\u003c/p\u003e \u003cp\u003e \u003cem\u003eColony formation assay.\u003c/em\u003e Transfected cells (2\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) were inoculated into 6-well plates and incubated with 10% FBS for 14 days. Then, colonies were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet dye (Beijing Solarbio Science \u0026amp; Technology Co., Ltd.) for 30 min. Colonies consisted of \u0026ge;\u0026thinsp;50 cells were counted and analyzed using Image J software (NIH).\u003c/p\u003e \u003cp\u003e \u003cem\u003eTranswell invasion and migration assays.\u003c/em\u003e A Transwell chamber (Corning; Corning, Inc.) was coated with 20 \u0026micro;l Matrigel (1 mg/ml; Beijing Solarbio Science \u0026amp; Technology Co., Ltd.), according to the manufacturer's instructions. For migration (without Matrigel) and invasion (with Matrigel; MilliporeSigma) assays, 4x10\u003csup\u003e4\u003c/sup\u003e BC cells were seeded into the upper chamber of the Transwell insert in 200 \u0026micro;l FBS-free medium. The lower chamber was supplemented with 600 \u0026micro;l 10% FBS medium as a chemoattractant. Following incubation at 37˚C for the indicated time points, non-invading cells were removed using a cotton swab. Cells adhering to the bottom surface of the membrane were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet for 20 min. The stained cells were counted using ImageJ software (NIH).\u003c/p\u003e \u003cp\u003e \u003cem\u003eWound healing assay.\u003c/em\u003e Transfected cells were cultured in 6-well plates until reached 100% confluency. Subsequently, the cell monolayer was scraped linearly using a 10-\u0026micro;l pipette tip prior rinsing away any isolated cells with PBS. Subsequently, the remaining cells were cultured in FBS-free medium and then the artificial wound closure was observed. Images of the migrated cells were captured at 0, 24 or 48 h. The width of the wound area was measured by ImageJ software (NIH) and the mobility ratio was determined using the following formula: Mobility=(scratch width at 0 h-scratch width at 24 or 48 h)/scratch width at 0 h x100%.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLuciferase reporter assay.\u003c/em\u003e The wild type (WT) and mutant (MUT) lncRNA DDX11-AS1 sequences and the 3'-untranslated regions (3'-UTR) of WT or MUT MTDH encompassing miR-30c-5p binding sites were inserted into the GP-miRGLO luciferase vector (Shanghai GenePharma Co., Ltd.). HEK293T cells were co-transfected with miR-30c-5p or miR-NC mimics and luciferase reporter plasmids for 48 h at 37˚C using the FuGENE\u0026reg; HD Transfection Reagent (Promega Corporation). Cells were harvested and the luciferase activity was measured using a Dual-luciferase reporter gene assay kit (Shanghai GenePharma Co., Ltd.), according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWestern blot assay.\u003c/em\u003e Cell lysis and protein separation was carried out using a RIPA lysis buffer (Beijing Solarbio Science \u0026amp; Technology Co., Ltd.) supplemented with protease and phosphatase inhibitor intact tablets (Beijing Solarbio Science \u0026amp; Technology Co., Ltd.). The protein concentration was measured using a BCA Protein Assay kit (Wuhan Boster Biological Technology, Ltd.). Total proteins were separated by 10% SDS-PAGE and were then electrotransfered onto PVDF membranes. Following blocking with 5% skimmed milk powder for 2 h at room temperature, the PVDF membranes were then incubated with primary antibodies against MTDH (dilution, 1:1000; rabbit; cat. no. 13860-1-AP), IκBα (dilution, 1:1000; rabbit; cat. no. 10268-1-AP), phosphorylation of IκBα (p-IκBα) (dilution, 1:5000; rabbit; cat. no. 82349-1-RR), p65 (dilution, 1:2000; rabbit; cat. no.10745-1-AP), p-p65 (dilution, 1:2000; rabbit; cat. no.82335-1-RR), Lamin B1 (dilution, 1:1000; rabbit; cat. no.12987-1-AP), and GAPDH (dilution, 1:10000; rabbit; cat. no.10494-1-AP; all from Proteintech Group, Inc.) at 4˚C overnight. The following day, the membranes were washed thrice with TBS-Tween for 10 min and were then incubated with the corresponding secondary antibodies at room temperature for 2 h. Finally, the protein bands were visualized using the Chemidoc XRS imaging system (Bio-Rad Laboratories) with an ECL solution.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical analysis.\u003c/em\u003e All statistical analyses were performed using the SPSS 26.0 (IBM Corp.) and GraphPad Prism 8 (GraphPad Software, Inc.) software. All measured data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The differences among multiple groups were compared by one-way ANOVA and Tukey's post hoc test or χ\u003csup\u003e2\u003c/sup\u003e test. Pearson correlation analysis was carried out to assess the association between DDX11-AS1 and miR-30c-5p in cell lines. All experiments were performed in at least three independent replicates. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eDDX11-AS1 is upregulated in BC\u003c/em\u003e. To predict the key lncRNAs, which could be involved in BC progression, the public microarray GEO dataset GSE156229 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE156229\u003c/span\u003e\u003c/span\u003e), containing the differential lncRNA expression profiles between tumor and normal tissues from patients with BC were analyzed (fold change, \u0026ge;\u0026thinsp;2 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). The analysis predicted that lncRNA DDX11-AS1 was significantly differentially expressed in BC tissues. RNA-seq data from a BC cohort in the TCGA database also revealed that DDX11-AS1 expression was higher in BC tissues compared with normal breast tissues, thus suggesting that DDX11-AS1 could promote the progression of BC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). In addition, RT-qPCR analysis showed that DDX11-AS1 was notably upregulated in BC tissues derived from 36 patients with BC compared with normal tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). The clinicopathological characteristics of patients with BC are listed in Table\u0026nbsp;1. Furthermore, higher DDX11-AS1 expression levels were associated with advanced-stage (III) and lymph node-metastasis in patients with BC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee and f). Simultaneously, compared with MCF10A normal breast epithelial cells, DDX11-AS1 expression levels were significantly enhanced in the BT549, MDA-MB-231, MCF7, MDA-MB-453 and SK-BR3 BC cell lines. The results showed the highest expression in SK-BR3 cell line and slightly lower expression in BT549 and MDA-MB-231, but the difference was not significant. In addition, we verified that both BT549 and MDA-MB-231 functional results were statistically significant by pre-experiment, but the difference of MDA-MB-231 was more significant, so we selected these two cell lines for the next validation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Collectively, the above findings indicated that the DDX11-AS1 expression levels were increased in BC tissues and BC cell lines, and it could play a potential carcinogenic role in BC progression.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDDX11-AS1 promotes BC cell proliferation, colony formation, migration and invasion.\u003c/em\u003e To evaluate the particular effects of DDX11-AS1 on BC, MDA-MB-231 cells were transfected with the pcDNA3.1 overexpression plasmid encompassing the DDX11-AS1 sequence. In addition, SK-BR3 cells were transfected with siRNA clones targeting DDX11-AS1 (si-DDX11-AS1-1,2,3). The above two cell lines were the most efficiently transfected ones. RT-qPCR analysis showed that DDX11-AS1 was successfully upregulated in MDA-MB-231 cells and downregulated in SK-BR3 cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea and c). Since si-DDX11-AS1-2 exhibited the most potent silencing activity, this siRNA clone was used for the subsequent functional experiments. Therefore, CCK-8 assays demonstrated that SK-BR3 cell transfection with si-DDX11-AS1-2 significantly inhibited cell proliferation, while DDX11-AS1 overexpression in MDA-MB-231 cells showed the opposite effect (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb and d). Consistently, clone formation assays further verified that DDX11-AS1 knockdown significantly inhibited SK-BR3 BC cell proliferation, while DDX11-AS1 overexpression significantly promoted the colony formation ability of MDA-MB-231 cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee-f). Furthermore, wound healing and Transwell assays were performed to assess the effects of DDX11-AS1 on the invasion and migration abilities of BC cells. The results revealed that DDX11-AS1 overexpression in MDA-MB-231 cells promoted cell invasion and migration, while SK-BR3 cell transfection with si-DDX11-AS1-2 yielded the opposite results (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg-j).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDDX11-AS1 sponges miR-30c-5p in BC cells\u003c/em\u003e. It is widely accepted that the functions and mechanisms of lncRNAs are associated with their particular cellular localization [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, a subcellular distribution assay was performed to verify the subcellular localization of DDX11-AS1 in BC cells. DDX11-AS1 could be detected in both the nuclear and cytoplasmatic fractions of MDA-MB-231 and SK-BR3 cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). It was therefore hypothesized that DDX11-AS1 could act as a ceRNA to sponge miRNAs. To further verify the mechanism underlying the effect of DDX11-AS1 on BC, the expression profile of miRNAs was analyzed using the GEO GSE156229 database (Fold change, \u0026le;-2 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). Additionally, the StarBase V3.0 database predicted that DDX11-AS1 encompassed binding sites for both miR-30c-5p and miR-30e-5p. In addition, bioinformatics analysis revealed that miR-30c-5p was significantly downregulated in the GEO GSE156229 dataset. However, the expression levels of miR-30e-5p were higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed and e). Additionally, the expression levels of miR-30c-5p were decreased in BC tissues compared with normal tissues. The above finding was consistent with that observed in the GEO dataset (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef). To verify the results provided by the bioinformatics analysis, DDX11-AS1 was silenced in SK-BR3 cells and overexpressed in MDA-MB-231 cells. RT-qPCR analysis showed that miR-30c-5p was upregulated in DDX11-AS1-depleted BC cells and downregulated in DDX11-AS1 overexpressing BC cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg-h). The above finding verified that miR-30c-5p could be a potential target of DDX11-AS1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ei). Subsequently, dual-luciferase reporter gene assays were performed in 293T cells to confirm whether DDX11-AS1 could sponge miR-30c-5p. Therefore, the luciferase activity of the DDX11-AS1-WT reporter plasmid was significantly reduced in cells co-transfected with miR-30c-5p mimics, while this inhibition was eliminated when the binding site of miR-30c-5p in DDX11-AS1 sequence was mutated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ej). The aforementioned results verified that DDX11-AS1 could sponge miR-30c-5p to inhibit its expression.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003emiR-30c-5p silencing abolishes the suppressive effect of DDX11-AS1 knockdown on BC cells.\u003c/em\u003e Subsequently, rescue experiments were carried out to further investigate whether DDX11-AS1 could promote the progression of BC via regulating miR-30c-5p. CCK-8 and colony formation assays demonstrated that miR-30c-5p silencing partially reversed the inhibitory effect of DDX11-AS1-2 silencing on cell proliferation, while miR-30c-5p overexpression could rescue the effect of DDX11-AS1 overexpression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea-f). Additionally, the wound healing assays displayed the same results (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). In general, the above findings suggested that miR-30c-5p could be an inhibitory target of DDX11-AS1 in BC.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDDX11-AS1 sponges miR-30c-5p to regulate the expression of MTDH in BC cells\u003c/em\u003e. Bioinformatics analysis using the miRDB, miRWalk, miRanda and TargetScan databases predicted that MTDH could be a potential target of miR-30c-5p (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Furthermore, the analysis also revealed that MTDH expression was positively and negatively associated with that of DDX11-AS1 and miR-30c-5p, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec and d). In addition, the TargetScan database predicted that MTDH encompassed a sequence complementary to miR-30c-5p. This result suggested that MTDH could act as a potential target of miR-30c-5p and it was therefore selected for further investigation. Therefore, luciferase assays showed that miR-30c-5p overexpression reduced the luciferase activity of the WT MTDH reporter plasmid, but not that of the MUT reporter plasmid, thus further verifying that MTDH was a direct target of miR-30c-5p (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb and e). The effect of DDX11-AS1 on the protein expression levels of MTDH was then assessed by western blot analysis. The results demonstrated that the protein expression levels of MTDH were significantly enhanced by DDX11-AS1 overexpression and miR-30c-5p knockdown, while they were reduced by DDX11-AS1 knockdown and miR-30c-5p overexpression.(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef-i).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDDX11-AS1/miR-30c-5p/MTDH axis promotes the progression of BC via regulating the activation of the NF-\u0026kappa;B signaling pathway\u003c/em\u003e. To determine the role of the DDX11-AS1/miR-30c-5p/MTDH axis in BC, MDA-MB-231 cells were co-transfected with miR-30c-5p mimics with or without a MTDH overexpression plasmid. In addition, SK-BR3 cells were transfected with miR-30c-5p inhibitor with or without a siRNA clone targeting MTDH. The transfection efficiency was determined by RT-qPCR (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea and c). The results showed that miR-30c-5p overexpression inhibited BC cell proliferation and this effect was reversed by MTDH overexpression. On the contrary, transfection of SK-BR3 cells with miR-30c-5p inhibitors significantly promoted cell proliferation, which was relieved by MTDH knockdown (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb and d, e and f). Furthermore, miR-30c-5p overexpression attenuated BC cell migration and invasion, while miR-30c-5p knockdown enhanced the above processes. These effects were abolished by MTDH overexpression or knockdown, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg-j). In addition, western blot analysis demonstrated that overexpression of MTDH promoted ikba degradation, enhanced p-p65 to activate the NF-\u0026kappa;B pathway, and caused nuclear translocation of p65, while knockdown of MTDH had the opposite result. (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ek and l). The above data suggested that DDX11-AS1 exerted a cancer promotive effect on BC via downregulating MTDH through competitively binding to miR-30c-5p \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003elncRNA, as a non-coding RNA, was involved in a range of biological functions related to carcinogenesis and had therefore received increasing attention [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Accumulated evidence has indicated that lncRNAs located in the cytoplasm serve as ceRNAs to protect target mRNAs from repression, while they are involved in gene regulation at the post-transcriptional level [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The significant role of lncRNAs as ceRNAs has been previously verified in BC. Therefore, a previous study demonstrated that the hypoxia inducible factor-1/lncRNA BCRT1/miR-1303/polypyrimidine tract-binding protein 3 axis could further accelerate the progression of BC via promoting the exosome-mediated M2 macrophage polarization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, the lncRNA-MAFG-AS1/miR-339-5p/cyclin-dependent kinase 2 axis could promote the progression of ER\u0026thinsp;+\u0026thinsp;BC and the resistance of BC cells to tamoxifen [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Another study revealed that lncRNA-NRON inhibited BC development via regulating the miR-302b/serine- and arginine-rich splicing factor 2 axis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Other studies also demonstrated that DDX11-AS1 was upregulated in drug resistant BC cell lines, thus suggesting that it could be involved in chemotherapy resistance in BC [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nonetheless, the biological significance of DDX11-AS1 in BC remains largely unknown. To the best of our knowledge, the current study was the first to show that DDX11-AS1 was significantly upregulated in BC tissues. At the same time, the DDX11-AS1 expression levels were increased with higher pathological stage and lymph node metastasis. Functional studies demonstrated that DDX11-AS1 overexpression facilitated the proliferation, invasion and migration of BC cells \u003cem\u003ein vitro\u003c/em\u003e, thus supporting the tumor-promotive role of DDX11-AS1 in BC.\u003c/p\u003e \u003cp\u003ePrevious studies also indicated that uncontrolled NF-κB expression could be associated with the development of BC [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, the activation of NF-κB could inhibit apoptosis and promote metastasis, thereby providing BC with a more aggressive phenotype [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The present study revealed that DDX11-AS1 could promote BC progression via regulating the NF-κB signaling pathway.\u003c/p\u003e \u003cp\u003eHerein, subcellular distribution assays also showed that DDX11-AS1 was expressed in the cytoplasm of BC cells, thus suggesting that it could act as a ceRNA to sponge miRNAs. Previous studies demonstrated that miR-30c-5p could inhibit the progression of different types of cancer, such as oral squamous cell carcinoma, gastric cancer and clear cell renal cell carcinoma [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Pei \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] indicated that miR-30c-5p could restrain tumor metastasis and BC cell migration via targeting coactosin-like protein 1. Furthermore, Yen \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] demonstrated that isolinderalactone inhibited the STAT3 signaling pathway via regulating suppressor of cytokine signaling 3 and miR-30c in BC. Herein, bioinformatic analysis using the StarBase V3.0 database predicted that miR-30c-5p encompassed a complementary sequence of DDX11-AS1. This finding was further verified by dual-luciferase reporter assays. Additionally, the results showed that DDX11-AS1 was upregulated and miR-30c-5p was downregulated in BC tissues, further supporting that DDX11-AS1 expression was negatively associated with that of miR-30c-5p. Therefore, miR-30c-5p overexpression could partially reverse the promotive effect of DDX11-AS1 on the behavior of BC cells. Overall, the results suggested that DDX11-AS1 could sponge miR-30c-5p to inhibit its expression.\u003c/p\u003e \u003cp\u003eMTDH is ubiquitously expressed in almost all human tissues at variable levels, while its expression levels are dramatically enhanced in cancer [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. A study revealed that the abundance of MTDH was positively associated with advanced clinical stages, clinicopathological features, distant metastasis and poor survival in patients with BC [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. MTDH expression in cancer cells can be modulated through a miRNA-mediated post-translational process [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For example, miR-375 could attenuate the growth of hepatocellular carcinoma via downregulating MTDH [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, dual-luciferase reporter assays combined with bioinformatics analysis indicated that MTDH could be a direct target of miR-30c-5p. Besides, DDX11-AS1 positively regulated MTDH expression in BC. Furthermore, the effects of the DDX11-AS1/miR-30c-5p/MTDH axis in the progress of BC were verified by \u003cem\u003ein vitro\u003c/em\u003e rescue experiments, and our results revealed that overexpression of MTDH could activate the NF-κB pathway by enhancing p-p65 and caused nuclear translocation of p65, which is consistent with previous studies [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Previous studies showed that MTDH and NF-κB were involved in paclitaxel and doxorubicin drug resistance, and MTDH overexpression may mediate trastuzumab resistance by increasing p65 nuclear translocation [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, DDX11-AS1 was upregulated in drug resistant BC cell lines, thus suggesting that DDX11-AS1 could be involved in the resistance of BC cells to paclitaxel, doxorubicin and trastuzumab through the MTDH/NF-κB axis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. It was therefore hypothesized that the DDX11-AS1/miR-30c-5p/MTDH axis could serve a key role in the chemoresistance of BC cells. However, our study did not have the means to validate the correlation between DDX11-AS1 and the prognosis of BC patients due to the limitation of available specimens..\u003c/p\u003e \u003cp\u003eIn conclusion, the present study demonstrated that DDX11-AS1 acted as a tumor promoter factor in BC. As a sponge of miR-30c-5p, DDX11-AS1 weakened its inhibitory effect on MTDH and promoted cell proliferation, migration, invasion and the activation of the NF-κB signaling pathway in BC. Overall, the present study suggested that DDX11-AS1 could be a potential target in BC therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Provincial Health Commission of Hebei province (grant no. 20230485), the Natural Science Foundation of Hebei Province (grant nos. H2020206365 and H2021206071), the S\u0026amp;T Program of Hebei (grant no. 236Z7719G), the Special Fund for Clinical Research of the Wu Jieping Medical Foundation (grant no. 320.6750.2020/07/17) and the Bethune Cancer Basic Research Program (grant no. BCF-NH-ZL-20201119-013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZS conceived, designed and oversaw the study. SL collected the clinical specimens and data. YTL and MSZ conducted the experiments. YTL and MSZ analyzed and interpreted the data. YTL wrote the manuscript. ZS revised the manuscript critically for significant intellectual content. YTL and MSZ confirmed the authenticity of all raw data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of 2021KY056.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023. Ca-Cancer J Clin 73(1): 17-48.\u003c/li\u003e\n\u003cli\u003eSaad ED, Katz A, Buyse M (2010) Overall survival and post-progression survival in advanced breast cancer: a review of recent randomized clinical trials. J Clin Oncol 28(11): 1958-62.\u003c/li\u003e\n\u003cli\u003eLiu P, Wang ZH, Ou XQ, Wu P, Zhang Y, Wu S, et al(2022) The FUS/circEZH2/KLF5/ feedback loop contributes to CXCR4-induced liver metastasis of breast cancer by enhancing epithelial-mesenchymal transition. Mol Cancer 21(1): 198.\u003c/li\u003e\n\u003cli\u003eZeng Y, Du W, Huang ZY, Wu S, Ou XQ, Zhang JH, et al(2023) Hsa_circ_0060467 promotes breast cancer liver metastasis by complexing with eIF4A3 and sponging miR-1205. Cell Death Discov 9(1): 153.\u003c/li\u003e\n\u003cli\u003eChen Z, Chen X, Chen P, Yu S, Nie F, Lu B, et al (2017) Long non-coding RNA SNHG20 promotes non-small cell lung cancer cell proliferation and migration by epigenetically silencing of P21 expression. Cell Death Dis 8(10): e3092.\u003c/li\u003e\n\u003cli\u003eLi D, Cheng M, Niu Y, Chi X, Liu X, Fan J, et al (2017) Identification of a novel human long non-coding RNA that regulates hepatic lipid metabolism by inhibiting SREBP-1c. Int J Biol Sci 13(3): 349-357.\u003c/li\u003e\n\u003cli\u003ePrensner JR, Chinnaiyan AM (2011). The emergence of lncRNAs in cancer biology. Cancer Discov 1(5): 391-407.\u003c/li\u003e\n\u003cli\u003eSalmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP (2011) A ceRNA hypothesis: the rosetta stone of a hidden RNA language?. Cell 146(3): 353\u0026ndash;358.\u003c/li\u003e\n\u003cli\u003eParaskevopoulou MD, Hatzigeorgiou AG (2016) Analyzing MiRNA-LncRNA Interactions. Methods Mol Biol 1402: 271\u0026ndash;286.\u003c/li\u003e\n\u003cli\u003eAn Y, Furber KL, Ji S (2017) Pseudogenes regulate parental gene expression via ceRNA network. J Cell Mol Med 21(1): 185\u0026ndash;192.\u003c/li\u003e\n\u003cli\u003eLiang Y, Song X, Li Y, Chen B, Zhao W, Wang L, et al (2020) LncRNA BCRT1 promotes breast cancer progression by targeting miR-1303/PTBP3 axis. Mol Cancer 19(1): 85.\u003c/li\u003e\n\u003cli\u003eGao J, Yuan Y, Zhang L, Yu S, Lu J, Feng J, et al (2020) Inhibition of ZEB1-AS1 confers cisplatin sensitivity in breast cancer by promoting microRNA-129-5p-dependent ZEB1 downregulation. Cancer Cell Int 20: 90.\u003c/li\u003e\n\u003cli\u003eWan T, Zheng J, Yao R, Yang S, Zheng W, Zhou P (2021) LncRNA DDX11-AS1 accelerates hepatocellular carcinoma progression via the miR-195-5p/MACC1 pathway. Ann Hepatol 20: 100258.\u003c/li\u003e\n\u003cli\u003eLiu J, Yang X, Gao S, Wen M, Yu Qiong (2023) DDX11-AS1 modulates DNA damage repair to enhance paclitaxel resistance of lung adenocarcinoma cells. Pharmacogenomics 24(3): 163-172.\u003c/li\u003e\n\u003cli\u003eMarchese FP, Grossi E, Mar\u0026iacute;n-B\u0026eacute;jar O, Bharti SK, Raimondi I, Gonz\u0026aacute;lez J, et al (2016) A long noncoding RNA regulates sister chromatid cohesion. Mol Cell 63(3): 397\u0026ndash;407.\u003c/li\u003e\n\u003cli\u003eSi X, Zhang G, Li M, Yao M, Shi X, Dong Z, et al (2023) DDX11-AS1 Promotes Chemoresistance through LIN28A-Mediated ATG12 mRNA Stabilization in Breast Cancer. PHARMACOLOGY 108(1): 61-73.\u003c/li\u003e\n\u003cli\u003eLiang M, Zhu B, Wang M, Jin J (2022) Knockdown of long non‑coding RNA DDX11‑AS1 inhibits the proliferation, migration and paclitaxel resistance of breast cancer cells by upregulating microRNA‑497 expression. Mol Med Rep 25(4): 123.\u003c/li\u003e\n\u003cli\u003eYang L, Tian Y, Leong WS, Song H, Yang W, Wang M, et al (2018) Efficient and tumor-specific knockdown of MTDH gene attenuates paclitaxel resistance of breast cancer cells both in vivo and in vitro. Breast Cancer Res 20(1): 113.\u003c/li\u003e\n\u003cli\u003eLi J, Zhang N, Song LB, Liao WT, Jiang LL, Gong LY, et al (2008) Astrocyte elevated gene-1 is a novel prognostic marker for breast cancer progression and overall patient survival. Clin Cancer Res 14(11): 3319-26.\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(\u0026minus;Delta Delta C(T)) method. Methods 25(4): 402\u0026ndash;408.\u003c/li\u003e\n\u003cli\u003eZhang K, Shi Z, Chang Y, Hu Z, Qi H, Hong W (2014) The ways of action of long non-coding RNAs in cytoplasm and nucleus. Gene 547(1): 1-9. \u003c/li\u003e\n\u003cli\u003eSun M, Nie F, Wang Z, De W (2016) Involvement of lncRNA dysregulation in gastric cancer. Histol And Histopathol 31(1): 33\u0026ndash;39.\u003c/li\u003e\n\u003cli\u003eCen SZ, Peng XJ, Deng JW, Jin HY, Deng ZN, Lin XH, et al (2023) The role of AFAP1-AS1 in mitotic catastrophe and metastasis of triple-negative breast cancer cells by activating the PLK1 signaling pathway. Oncol Res 31(3): 375-388.\u003c/li\u003e\n\u003cli\u003eFeng J, Wen T, L Z, Feng L, Zhou L, Yang Z, et al (2020) Cross-talk between the ER pathway and the lncRNA MAFG-AS1/miR-339-5p/ CDK2 axis promotes progression of ER+ breast cancer and confers tamoxifen resistance. Aging (Albany NY) 12(20): 20658-20683.\u003c/li\u003e\n\u003cli\u003eMao Q, Li L, Zhang C, Sun Y, Liu S, Li Y, et al (2020) Long non coding RNA NRON inhibited breast cancer development through regulating miR-302b/SRSF2 axis. Am J Transl Res 12(8): 4683-4692.\u003c/li\u003e\n\u003cli\u003eRomieu-Mourez R, Kim DW, Shin SM, Demicco EG, Landesman-Bollag E, Seldin DC, et al (2003) Mouse mammary tumor virus c-rel transgenic mice develop mammary tumors. Mol Med Rep 23(16): 5738-5754.\u003c/li\u003e\n\u003cli\u003eZhou J, Hao Z, Gu P , Bai J, Margolick JB, Zhang Y (2008) NF-kappaB pathway inhibitors preferentially inhibit breast cancer stem-like cells. Breast Cancer Res Tr 111(3): 419-427.\u003c/li\u003e\n\u003cli\u003eWu JT, Kral JG (2005) The NF-kappaB/IkappaB signaling system: a molecular target in breast cancer therapy. J Surg Res 123(1): 158-169.\u003c/li\u003e\n\u003cli\u003eMehterov N, Vladimirov B, Sacconi A, Pulito C, Rucinski M, Blandino G, et al (2021) Salivary miR-30c-5p as Potential Biomarker for Detection of Oral Squamous Cell Carcinoma. Biomedicines 9(9): 1079.\u003c/li\u003e\n\u003cli\u003eCao J, Li G, Han M, Xu H, Huang K (2017) MiR-30c-5p suppresses migration, invasion and epithelial to mesenchymal transition of gastric cancer via targeting MTA1. Biomed Pharmacother 93: 554-560.\u003c/li\u003e\n\u003cli\u003eOuteiro-Pinho G, Barros-Silva D, Moreira-Silva F, Lobo J, Carneiro I, Morais A, et al (2022) Epigenetically-regulated miR-30a/c-5p directly target TWF1 and hamper ccRCC cell aggressiveness. Transl Res 249: 110-127.\u003c/li\u003e\n\u003cli\u003ePei B, Li T, Qian Q, Fan W, He X, Zhu Y, et al (2020) Downregulation of microRNA-30c-5p was responsible for cell migration and tumor metastasis via COTL1-mediated microfilament arrangement in breast cancer. Gland Surg 9(3): 747-758.\u003c/li\u003e\n\u003cli\u003eYen MC, Shih YC, Hsu YL, Lin ES, Lin YS, Tsai EM, et al (2016) Isolinderalactone enhances the inhibition of SOCS3 on STAT3 activity by decreasing miR-30c in breast cancer. Oncol Rep 35(3): 1356-64.\u003c/li\u003e\n\u003cli\u003eKang DC, Su ZZ, Sarkar D, Emdad L, Volsky DJ, Fisher PB (2005) Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1. Gene 353(1): 8\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eWan L, Kang Y (2013) Pleiotropic roles of AEG-1/MTDH/LYRIC in breast cancer. Adv Cancer Res 120: 113-134.\u003c/li\u003e\n\u003cli\u003eKhan M, Sarkar D (2021) The scope of astrocyte elevated Gene-1/Metad-herin (AEG-1/MTDH) in Cancer Clinicopathology: A Review. Genes (Basel) 12(2): 308.\u003c/li\u003e\n\u003cli\u003eHe X, Chang Y, Meng F, Wang M, Xie Q, Tang F, et al (2012) MicroRNA-375 targets AEG-1 in hepatocellular carcinoma and suppresses liver cancer cell growth in vitro and in vivo. Oncogene 31(28): 3357\u0026ndash;3369.\u003c/li\u003e\n\u003cli\u003eDu C, Yi X, Liu W, Han T, Liu Z, Ding Z, et al (2014) MTDH mediates trastuzumab resistance in HER2 positive breast cancer by decreasing PTEN expression through an NF-\u0026kappa;B-dependent pathway. BMC Cancer 14: 869.\u003c/li\u003e\n\u003cli\u003eSong Z, Wang Y, Li C, Zhang D, Wang X (2015) Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin. Plos One 10(5): e0127599. \u003c/li\u003e\n\u003cli\u003eAbdin SM, Tolba MF, Zaher DM, Omar HA (2021) Nuclear factor-\u0026kappa;B signaling inhibitors revert multidrug-resistance in breast cancer cells. Chem-biol Interact 340: 109450. \u003c/li\u003e\n\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":"DDX11-AS1, miR-30c-5p, metadherin, breast cancer, progression","lastPublishedDoi":"10.21203/rs.3.rs-3822928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3822928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLong non-coding RNAs (lncRNAs) serve a significant role in the occurrence and development of malignant tumors. However, the roles of lncRNAs in breast cancer (BC) remain largely unknown. Therefore, the current study aimed to investigate the effect of lncRNA DDX11-AS1 on BC progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioinformatics analysis using public microarray revealed that DDX11-AS1 was upregulated in BC. In addition, the effect of DDX11-AS1 on the prognosis of patients with BC was evaluated by clinical data analysis. Furthermore, the proliferation, migration and invasion abilities of BC cells were assessed in vitro in the MDA-MB-231 and SK-BR3 BC cell lines. Luciferase reporter assay was carried out to unveil the association between DDX11-AS1, microRNA (miR)-30c-5p and metadherin (MTDH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDDX11-AS1 was significantly upregulated in BC tissues and cells. Additionally, bioinformatics analysis revealed that the expression levels of DDX11-AS1 were increased with enhanced pathological grading and lymph node metastasis. Furthermore, DDX11-AS1 knockdown markedly inhibited the proliferation, migration and invasion abilities of BC cells. Mechanistically, DDX11-AS1 could prevent the degradation of MTDH in BC via competitively binding with miR-30c-5p, which could act as a tumor promoter factor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollectively, the above results suggested that the DDX11-AS1/miR-30c-5p/MTDH axis could be associated with the progression of BC and DDX11-AS1 could be a potential biomarker and therapeutic target for BC.\u003c/p\u003e","manuscriptTitle":"LncRNA DDX11-AS1 promotes breast cancer progression via targeting the miR-30c-5p/MTDH axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 17:27:17","doi":"10.21203/rs.3.rs-3822928/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":"4b1998c5-6262-41e9-a8ff-d56951fae771","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-05T13:59:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-03 17:27:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3822928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3822928","identity":"rs-3822928","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}