The Roles And Mechanisms Of LINC00301,LINC00511 And XIST In Non-small Cell Lung Cancer

Lung cancer is one of the leading malignant tumors in human.In recent years,with the increasing environmental pollution,the incidence rate will continue and show a significant upward trend in the follow-up period.China is a high incidence of lung cancer in the world,nearly a decade of lung cancer has accounted for male and female malignant tumor incidence of the first and second,including non-small cell lung cancer(NSCLC)accounted for more than 80%of all cases of lung cancer,and most patients have been diagnosed when the late loss of surgical opportunities.The pathogenesis of lung cancer is unclear,and its effective early diagnosis and prevention methods are lacked.Over the years,domestic and foreign scholars from different angles try to explore the mechanism of lung cancer,and find that cancer cells and normal cells are different in the proliferation capacity,apoptosis level,gene mutation rate,resistance capacity.The occurrence and development of lung cancer involved a variety of cancer genes overexpression,and tumor suppressor genes inactivation.But the precise molecular mechanism for its development is not yet fully understood.Therefore,further to explore the relationship between the change of new gene function and the development of lung cancer and its malignant features,and to find out the molecular mechanism of early diagnosis and prognosis,especially in the blood detection,to design reasonable drugs,to further improve the diagnosis and treatment of lung cancer in China,are of great significance.Long non-coding RNA(IncRNA)is a class of RNA molecules that transcribe more than 200 nt(nucleotides),lacking the function of coding proteins.It is located in the nucleus or cytoplasm,and involved in transcriptional regulation and post-transcriptional regulation,and so on,through regulating gene expression levels.The transcripts of 4%to 9%of the human genome sequence are lncRNAs,many of which appear only at specific developmental stages,with tissue or cell specificity.LncRNA can bind to specific proteins,inhibit mRNA degradation,produce internal siRNAs,regulate protein activity,alter protein localization,and so on.It is involed in transcriptional interference,chromatin remodeling and histone modification,hybridization for antisense and sense strand RNA.Different levels of regulation associated with the protein coding genes are involved in the induction of disease.These findings open up a new field of research on the mechanism of lncRNA in etiology.This study was divided into three parts.The first part of the study showed that LINC00301 was highly expressed in NSCLC by analyzing the differential expression of IncRNA in lung cancer tissues.Further studies have shown that the expression of LINC00301 is significantly higher in NSCLC tissues and cells.pcDNA3.1-LINC00301 and sh-LINC00301 were transfected into human lung cancer cell line A549 and SPC-A-1 cells by liposome 2000.Fluorescence in situ hybridization(FISH)was used to detect the localization of LINC00301 in cells.The effect of LINC00301 on the proliferation of lung cancer cells was detected by trypan blue staining,CCK-8,clone formation assay and EdU proliferation assay.The transwell migration experiment was used to study LINC00301.The role of LINC00301 on the growth of lung cancer cells was studied.The effect of LINC00301 on the methylation of lung cancer cell lines A549 and SPC-A-1 cells was studied.The effect of LINC00301 on the methylation of lung cancer cells was studied.The interaction between LINC00301 and EZH2 was detected by western blot,qRT-PCR and ChIP.The effect of LINC00301 and EZH2 on the expression of EAF2 and its downstream HIF1? signaling pathway was predicted by RPISeq and catRAPID,and detected by RIP,RNA-pull down and EMSA.The effect of EAF2 on the proliferation of NSCLC cells was investigated by trypan blue staining and colony formation assay.The invasion of NSCLC cells was observed by transwell migration assay.Western blot was used to detect the expression of HIF1? in NSCLC tissues and cells.K-M plotter was used to detect the expression of HIF1? in NSCLC patients' survival.The relationship between LINC00301 and miR-1276 was predicted using bioinformatics software miRDB and verified by RNAi-pull down and luciferase reporter assay.The bioinformatics software TargetScan,PicTar,microRNA.org were used to predict,western blot and qRT-PCR were used to comfirm HIF1? was a direct target of miR-1276.LINC00301/miR-1276/HIF1? pathway was examined by western blot,trypan blue staining,colony formation assay and transwell migration/invasion assay in NSCLC cells.The results showed that LINC00301 was highly expressed in NSCLC tissues and cells and was closely related to the prognosis of NSCLC patients.LINC00301 promoted the proliferation,migration and invasion of NSCLC cells in vitro and promoted the growth of NSCLC cells.FISH showed that LINC00301 in the nucleus.LINC00301 specifically binds to EZH2,mediates the methylation of H3K27me3 at the promoter of EAF2 gene promoter,and inhibits the expression of EAF2,suppresses VHL phosphorylation and represses the hydrolysis of HIFla.HIF1? is highly expressed in NSCLC patients.The repression of miR-1276 to HIF1?mRNA 3'-UTR was released by binding to LLINC00301,and promoted the expression of HIF1? protein.The results suggest that LINC00301 can function as an oncogene for lung cancer by regulating the HIF1? signaling pathway.In the second part,we analyzed the differential expression of lncRNA in lung cancer tissues and found that LINC00511 was highly expressed in NSCLC.Further study found that the expression of LINC00511 in NSCLC tissue and cells was significantly increased.Transfection of sh-LINC00511 into human lung cancer cell A549 and SPC-A-1 cells by liposome 2000.The effect of LINC00511 on the proliferation of lung cancer cells was detected by CCK-8,colony formation assay and EdU proliferation assay.The cell cycle of LINC00511 was detected by flow cytometry.The effect of LINC00511 on the migration ability of lung cancer cells was studied by transwell migration assay The effect of LINC00511 on the apoptosis of NSCLC cells was studied by flow cytometry,Hochest staining and caspase 3/7 activity.The effect of LINC00511 on the growth of lung cancer cells was studied by using the tumor formation model of nude mice.Western blot,qRT-PCR and ChIP were used to detect the interaction between LINC00511 and EZH2 and the regulation of p57.The role of p57 in LINC00301' oncogenic effects was evaluated by colony formation assay,EdU experiment,transwell migration and invasion assay,and flow cytometry in NSCLC cells.K-M plotter was used to analyze the prognosis value of p57 in NSCLC patients.The results showed that LINC00511 was highly expressed in NSCLC tissues and cells and was closely related to the prognosis of NSCLC patients.LINC00511 promoted the proliferation,migration and invasion of NSCLC cells in vitro,inhibited the apoptosis of NSCLC cells in vitro and promoted the expression of NSCLC cells.LINC00511 specifically binds to EZH2 to promote the methylation of H3K27me3 at the promoter of p57 gene and suppresses the expression of p57.p57 is low expressed in NSCLC and is closely related to the prognosis of NSCLC patients.The results suggest that LINC00511 can regulate the p57 signaling pathway as an oncogene in NSCLC.In the third part,we analyzed the differential expression of lncRNA in lung cancer tissues and found that XIST was highly expressed in NSCLC.Further studies have shown that XIST expression is significantly higher in NSCLC tissues and cells.Transfection of sh-XIST into human lung cancer cell line A549 and SPC-A-1 cells by liposome 2000.The effect of XIST on the proliferation of lung cancer cells was detected by colony formation assay.The cell cycle of lung cancer was detected by flow cytometry.The effect of XIST on the migration ability of lung cancer cells was studied by transwell migration assay.The interaction between XIST and EZH2 and the regulation of KLF2 were detected by RIP,RNA-pull down,western blot,qRT-PCR and ChIP.The colony formation experiment,transwell migration and invasion experiments,flow cytometry were performed to detect the role of KLF2 in the regulation of XIST's oncogenic effects on NSCLC cells.The results showed that XIST was highly expressed in NSCLC tissues and cells and was closely related to the prognosis of NSCLC patients.XIST promoted the proliferation,migration and invasion of NSCLC cells in vitro and promoted the growth of NSCLC cells.XIST was mainly located in the nucleus.XIST and EZH2 specifically promoted the methylation of H3K27me3 at the promoter of KLF2 gene and inhibited the expression of KLF2.KLF2 was low in NSCLC and was closely related to the prognosis of NSCLC patients.The results suggest that XIST can function as a cancer-promoting gene for NSCLC by regulating the KLF2 signaling pathway.