Mechanisms Of Long Non-coding RNA XIST Promoting TGF-β Induced Epithelial-mesenchymal Transition In Non-small Cell Lung Cancer

Background and Objective: Lung cancer is one of the most common malignances both in China and worldwide.Most of the lung cancer cases(~85%)belong to non-small cell lung cancer(NSCLC).The prognosis for advanced NSCLC patients is still poor due to tumor metastasis;therefore it is very important to elucidate the molecular mechanisms underlying NSCLC invasion and metastasis.TGF-β induced epithelial-mesenchymal transition(EMT)is proved to play a pivotal role in the invasion process of NSCLC cells,while the TGF-β signal pathway was reported to be regulated by many factors during NSCLC progression.In recent years,long non-coding RNA(lncRNA)and its role in regulation of gene expression have fascinated researchers to focus special lncRNAs’ functions on cancer progression.X inactivate-specific transcript(XIST)is found abnormally expression in several kinds of malignancies and associated with cancer progression.Recent studies showed that XIST is upregulated in NSCLC and played roles in NSCLC cells invasion and metastasis.In this study,we sought to elucidate the mechanisms of XIST promoting TGF-β induced EMT in NSCLC,and furthermore to supply new sights into NSCLC diagnosis and treatment.Methods:(1)The expression levels of lncRNA XIST and ZEB2 mRNA in NSCLC tissues with different metastatic status were detected by quantitative real-time PCR(qPCR).We also used qPCR to detect the expression levels of miR-367-3p,miR-141-3p and ZEB2 mRNA in different NSCLC cell lines and normal bronchial epithelial cells(HBE).(2)The expression levels of E-cadherin,N-cadherin and Vimentin,which were key markers of TGF-β induced EMT,were determined by western blot analysis afterXIST or ZEB2 knocking down,or miR-367-3p /miR-141-3p overexpression.(3)Using target prediction programs(TargetScan7.1 and starBase v2.0)we have found mi RNAs which were predicted to target both XIST and ZEB2.After that we constructed a series of luciferase reporter plasmids and luciferase assays were performed to detect whether the predicted miRNAs could directly binding to the objective lncRNA XIST and ZEB2 mRNA.(4)Anti-AGO2 RIP assays were conducted in A549 and H226 cells to determine whether miR-367 and miR-141 could regulate lncRNA XIST expression in an AGO2-dependent manner.RT-PCR was used to detect the expression of lncRNA XIST enriched by AGO2 pull-down.(5)LncRNA XIST-silencing stable A549 cells were constructed,and the effect of lncRNA XIST knockdown on NSCLC cells metastasis in vivo was detected.Results:(1)The expression levels of lncRNA XIST and ZEB2 mRNA were detected up-regulated in lymph node metastatic NSCLC tissues compared with non-metastatic tissues.(2)The expression levels of miR-367-3p or miR-141-3p were detected down-regulated in NSCLC cells compared with normal human bronchial epithelial cell line(HBE).Furthermore,the expression levels of these 2 mi RNAs were detected higher in highly metastatic 95 D cells than its lowly metastatic counterpart,95 C cells.The expression levels of ZEB2 mRNA were detected increased in NSCLC cell lines.(3)TGF-β induced EMT process could be significantly inhibited by knocking down of XIST.(4)2 miRNAs,miR-367-3p and miR-141-3p,were predicted by target prediction programs to target both XIST and ZEB2.The results of duel luciferase assay demonstrated that mi R-367-3p and mi R-141-3p could directly interact with lncRNA XIST and ZEB2 mRNA 3’-UTR.The results of Anti-AGO2 RIP assay confirmed that lncRNA XIST is a direct target of both miR-367-3p and miR-141-3p.Western blot assay results showed that the expression level of ZEB2 could be repressed obviously by over expression of mi R-367-3p or miR-141-3p in NSCLC cells.(5)The TGF-β induced EMT process in NSCLC could be repressed by ZEB2 knocking down.(6)MiR-367-3p or mi R-141-3p could repress TGF-β induced EMT process in NSCLC by targeting ZEB2.(7)The in vivo experiment of metastasis showed that knocking down of lncRNAXIST inhibits pulmonary metastasis of NSCLC cells.Conclusion: Our study reveals that lncRNA XIST could promote TGF-β induced EMT process and cell invasion and cancer metastasis in NSCLC by regulating mi R-367/miR-141-ZEB2 axis.