The Long Non-coding RNA XIST Controls Non-small Cell Lung Cancer Proliferation And Invasion By Modulating MiR-186-5p

Objective:Non-small cell lung cancer(NSCLC)accounts for approximately 80% of all lung cancers,which are the primary cause of cancer-related deaths worldwide.Despite advances in the early diagnosis and treatment of lung cancer,the prognosis of lung cancer patients remain poor,with the five-year overall survival rates currently at 18%.Hence,an improved and detailed understanding of the molecular mechanisms underlying NSCLC progression has important clinical significance.Recently,increasing evidence has shown that long non-coding RNAs(lncRNAs)provide new insights into the treatment of NSCLC pathogenesis.Protein-coding genes account for only 2% of the human genome,whereas the vast majority of transcripts are non-coding RNAs(ncRNAs),including lncRNAs and microRNA(miRNAs).A number of microRNAs were found to act as oncogenes or tumor suppressor genes by previous reports,whereas the mechanism of action for long ncRNAs(lncRNAs)remains largely unknown.LncRNAs are a class of non-coding RNA greater than 200 nucleotides with no protein-coding potential.LncRNAs play critical roles in numerous biological processes,such as epigenetic regulation,nuclear import,cell cycle control,imprinting,differentiation,alternative splicing,RNA decay and transcription.Recent studies have revealed that the expression of lncRNAs is frequently dysregulated in cancers including NSCLC,and is usually correlated to the long-term survival of patients.Further study found that lnc RNAs can be involved in the regulation of tumor cell proliferation,invasion,drug resistance and other malignant biological behavior.A complete understanding of the roles of lncRNAs in NSCLC would greatly advance the development of novel therapeutic strategies.The lncRNA XIST(X-inactive specific transcript),a product of the XIST gene,is the master regulator of X inactivation in mammals.XIST is highly expressed in some carcinomas including breast cancer,glioblastoma and ovarian cancer,suggesting that XIST may serve as a biomarker for the diagnosis of these cancers.A recent study showed that XIST is upregulated and is essential for the long term survival of NSCLC;however,its molecular role in NSCLC has not been clarified.Our study first confirmed that XIST expression was higher in human non-small cell lung cancer cell lines and tumor tissues than in human bronchial epithelial cell lines and paracancerous lung tissues.Next,we showed that XIST knockdown inhibited NSCLC proliferation and invasion in vitro and suppressed tumor growth in vivo.Finally,miR-186-5p was found to block the oncogenic functions of XIST in NSCLC cells.Therefore,our study identifies XIST as a novel therapeutic target for NSCLC treatment.Methods:1.Gene chip analysis the difference expression of the lncRNA XIST between the non-small cell lung cancer tissue and paracancerous tissue.2.Real-time PCR was used to examine the expression of lncRNA XIST in non-small cell lung cancer cell lines and tissues.3.Statistical analysis the 30 cases of non-small cell patients’ clinical pathology data.4.MTT assay was used to examine the activity of non-small cell lung cancer cells after knockdown the expression of XIST.5.Flow cytometry was used to examine the cell cycle and apoptosis of non-small cell lung cancer after knockdown the expression of XIST.6.Cell Migration Assay was used to examine the migration ability of non-small cell lung cancer cells after knockdown the expression of XIST.7.Transwell assay was used to examine the invasion ability of non-small cell lung cancer cells after knockdown the expression of XIST.8.Western blot was used to examine the non-small cell lung cancer cell proliferation invasion protein expression after knockdown the expression of XIST.9.Tumorigenicity test in nude mice to examine the growth ability of non-small cell lung cancer cells in vivo after knockdown the expression of XIST.10.Bioinformatics analysis the miRNAs that may bind to XIST.11.Build the XIST fragment vector with miR-186-5p binding site.12.Real-time PCR was used to examine the non-small cell lung cancer miR-186 expression levels after transfected siXIST.13.Luciferase reporter assay was used to examine whether XIST and miR-186 are binding.14.RIP experiments was used to examine whether XIST and miR-186 are presented in the same RISC complex.15.Rescue experiments was used to verify whether miR-186 can negatively regulate XIST.Results:1.The lncRNA XIST expression in non-small cell lung cancer cell lines and tissues are increased accompany with the prognosis of patients.We download the non-small cell lung cancer genome-wide microarray data chip GSE32867 information from pubmed GEO database and analyzed the lncRNA information contained in it.It was found that lncRNA XIST was one of the most significantly differentially expressed lncRNA and its expression level in tumor tissues was significantly higher than that in adjacent normal tissues.Subsequently,we further validated the differential expression in cancer tissues and paracancerous tissues of 30 non-small cell lung cancer patients who underwent the operation in the First Affiliated Hospital of China Medical University.Compared to normal lung tissue,the expression of XIST in non-small cell lung cancer tissues was significantly increased.In addition,we also examined the expression of XIST in different non-small cell lung cancer cell lines.Compared with the human immortalized bronchial epithelial cell line(BEAS-2B),the expression levels of XIST in the non-small cell lung cancer cell lines A549,H1299,Calu-3,and SK-MES-1 were significantly increased.These findings are consistent with the results in the chip GSE32867,suggesting that high expression of XIST may be involved in the occurrence and development of non-small cell lung cancer.To summarize the clinical pathology data of 30 patients,it is suggested that the expression level of XIST is related to gender,lymph node metastasis and TNM staging.Finding that high expression of XIST is more common in high-grade lung cancer,and the expression level of XIST may related to the prognosis of patients.2.Knockdown the expression of XIST inhibits the proliferation and invasiveness of non-small cell lung cancer cells.The optical density at 570 nm in siXIST group was significantly lower than that in siNC group in MTT assay.Knockdown the expression of XIST decreased the activity of A549 and H1299 cells.Flow cytometry results showed that G1 / G0-S phase arrest occurred in siXIST cells and the rate of early apoptosis was significantly increased.The results of Transwell suggest that the average number of cells per field of view in siXIST group decreased significantly compared with the control group.The results of Western blot suggested that the expression level of proliferation-invasion-related proteins in siXIST group decreased significantly.3.Knockdown the expression of XIST inhibits the growth of non-small cell lung cancer cells in vivo.The establishment of nude mice xenograft model to detect whether knockdown the XIST expression can affect tumor growth in vivo.We used LV-shXIST and LV-NC to construct stable knockdown XIST A549 cell lines and control cell lines,and subcutaneous injected into nude mice.Tumor size was measured after 30 days.We found that the tumor diameter and weight of LV-shXIST group were significantly smaller than those of LV-NC group.The growth rate of tumor after XIST knockdown was lower than that of negative control group.Tumor tissue sections of the LV-shXIST group showed weaker Ki67 staining compared to the control group,suggesting that knockdown the expression of XIST suppresses the growth of non-small cell lung cancer.4.XIST plays its biological role through binding to miR-186-5p in non-small cell lung cancer cells.Bioinformatics predict the mi R-186-5p is one of the most likely miRNAs to bind to XIST.The expression of miR-186-5p in siXIST group was detected by Real-time PCR,which indicated that the expression of miR-186-5p was significantly increased.Luciferase reporter gene results suggest that after up-regulation the expression of miR-186-5p in Wt group,the luciferase activity was significantly reduced,suggesting that XIST is a direct target of miR-186-5p.The results of RIP test showed that the expression levels of XIST and mi R-186-5p in Ago2 immune complexes were significantly higher than those in IgG,suggesting that XIST and miR-186-5p may exist in the same RISC complex in non-small cell lung cancer cells.5.LncRNA XIST regulates the proliferation and invasion of non-small cell lung cancer by regulating mi R-186-5p.Up-regulate the expression of miR-186-5p in A549 cells that had been transfected with XIST.MTT assay,flow cytometry and Transwell assay was used to examine the role of miR-186-5p in the regulation XIST of the proliferation and invasion in non-small cell lung cancer.MTT assay results showed that overexpression of XIST increased the proliferation of non-small cell lung cancer cells,overexpression of miR-186-5p inhibited the promotion of XIST in the proliferation of non-small cell lung cancer.Flow-through experiments showed that overexpression of XIST increased the proportion of S phase cells in non-small cell lung cancer cells and decreased the proportion of early apoptotic cells.Overexpression of miR-186-5p inhibited the effect of XIST on the cell cycle of NSCLC.Transwell experimental results suggest that overexpression of XIST increases the invasiveness of non-small cell lung cancer cells.Overexpression of miR-186-5p inhibits the promotion of XIST in invasive ability of non-small cell lung cancer.The above experimental results confirmed that XIST promotes the proliferation and invasion of non-small cell lung cancer by regulating the expression level of miR-186-5p.Conclusion: 1.The expression level of lncRNA XIST expression in non-small cell lung cancer cell lines and lung cancer patients was increased.2.The expression level of lncRNA XIST in non-small cell lung cancer tissues may correlated with the poor prognosis of patients.3.Knockdown the expression level of lncRNA XIST inhibits the proliferation and invasion of non-small cell lung cancer cells in vitro.4.Knockdown the expression level of lncRNA XIST inhibits the growth of non-small cell lung cancer cells in nude mice.5.Lnc RNAXIST binds directly to miR-186-5p in non-small cell lung cancer cells and may be present in the same RISC complex.6.lncRNA XIST promotes the proliferation and invasion of non-small cell lung cancer by regulating mi R-186-5p.