| Lung cancer is one of the leading causes of cancer-associated deaths worldwide,and non-small lung cancer(NSCLC)accounts for about 85% of all the total number of lung cancer cases.Despite the rapid development of lung cancer diagnosis and treatment in recent years,the 5-year survival rate is only about 15%.Therefore,it is urgent to understand the detailed molecular mechanism of the oncogenesis of lung cancer and look for diagnosis and prognosis markers of lung cancer.Recent studies have implied that lncRNA are related to the oncogenesis and development of many tpes of tumors,including lung cancer.In the previous work,we identified a lung cancer associated lncRNA OSER1-AS1 through bioinformatics pipelines.We explored the expression and prognostic significance of lncRNA OSER1-AS1 in NSCLC and further investigated the biological functions and potential mechanisms of lncRNA OSER1-AS1.Part ? The expression and prognostic significance of lncRNA OSER1-AS1 in NSCLC and the characterization of lncRNA OSER1-AS1.Objective: To explore the relative expression level of lncRNA OSER1-AS1 and its prognostic significance in NSCLC and to explore the characterization of lncRNA OSER1-AS1.Methods: The relative expression level of OSER1-AS1 in NSCLC tissues and adjacent normal tissues was detected by qRT-PCR.Survival analysis was conducted by online analysis at Kaplan-Meier plotter website.NCBI website and bioinformatics analysis were conducted to detect the genomic location and coding capability prediction of lncRNA OSER1-AS1.The subcellular localization of lncRNA OSER1-AS1 in lung cancer cells was detected by RNA FISH and qRT-PCR analysis.Results:1.The relative expression level of lncRNA OSER1-AS1 was significantly down-regulated in NSCLC tissues compared with adjacent normal tissues(P<0.01).2.Kaplan-Meier plot showed that low expression of lncRNA OSER1-AS1 in NSCLC was significantly associated with poor prognosis(P<0.01).3.lncRNA OSER1-AS1 was located at chromosomal 20q13.12,consisting of two exons with a full length of 1482 nt.LncRNA OSER1-AS1 was mainly located in the cytoplasm of lung cells,with no coding potential.Conclusion: LncRNA OSER1-AS1 expression was significantly down-regulated in NSCLC tissues and associated with poor prognosis in NSCLC.LncRNA OSER1-AS1 was located at chromosomal 20q13.12 and mainly located in the cytoplasm.Part ? The effects of lncRNA OSER1-AS1 on biological behavior of NSCLC cellsObjective: To explore the the effects of lncRNA OSER1-AS1 on the proliferation,migration and invasion of NSCLC cells.Methods: The siRNA and overexpression vectors were used to down-regulate and up-regulate the expression level of lncRNA OSER1-AS1 in NSCLC cells,respectively.The effects of OSER1-AS1 on proliferation,migration and invasion of NSCLC cells were detected by cell counting kit-8(CCK-8)assay and Transwell assay in vitro.Subcutaneous tumorigenesis assay and metastatic tumor experiment were used to examine the effects of lncRNA OSER1-AS1 in vivo.Results:1.The qRT-PCR showed that the expression level of lncRNA OSER1-AS1 was significantly down-regulated in NSCLC cells after transfected with siRNAs.The expression level of lncRNA OSER1-AS1 was significantly up-regulated in NSCLC cells after transfected with overexpression vectors.2.The cell behavior experiments showed that knockdown of lncRNA OSER1-AS1 promote proliferation,migration and invasion of H1299 and SPCA1 cells.On the contrary,the overexpression of lncRNA OSER1-AS1 resulted in the inhibition of proliferation,migration and invasion of H1299 and SPCA1 cells.3.Subcutaneous tumorigenesis assay showed that lncRNA OSER1-AS1 inhibited the growth of NSCLC cells in nude mice.Metastatic tumor experiment showed that lncRNA OSER1-AS1 inhibit the metastasis of NSCLC cells in Balb/c mice.Conclusion: lncRNA OSER1-AS1 can inhibit the growth and metastasis of NSCLC cells in vitro and in vivo,hence it may play a role of tumor-suppressor in oncogenesis and development of lung cancer.Part ? Transcription factor MYC regulates the expression of lncRNA OSER1-AS1Objective: To explore the potential mechanisms controlling the expression of lncRNA OSER1-AS1.Methods: UCSC genome browser was used to find transcription factor binding sites in lncRNA OSER1-AS1 promoter region.Chromatin Immunoprecipitation(ChIP)experiments and dual-luciferase reporter assays were performed to validate binding of MYC to lncRNA OSER1-AS1 promoter.The expression level of lncRNA OSER1-AS1 was detected by qRT-PCR after overexpression of MYC.Results:1.We found a binding site for MYC within the lncRNA OSER1-AS1 promoter region in UCSC genome browser.The result of ChIP experiments demonstrated the direct binding of MYC to lncRNA OSER1-AS1 promoter.2.Dual-luciferase reporter assays showed that overexpression of MYC can repressed the luciferase activity of pGL3-OSER1-AS1 vector.The expression level of lncRNA OSER1-AS1 was down-regulated after overexpression of MYC.Conclusion: Transcription factor MYC can down-regulate the expression of lncRNA OSER1-AS1.Part ? Molecular mechanisms of lncRNA OSER1-AS1 on the biological effect of NSCLC cellsObjective: To explore the molecular mechanisms by which lncRNA OSER1-AS1 contributed to the lung cancer progression.Methods: Bioinformatics analysis were conducted to seek miRNA and RNA binding protein(RBP)binding to lncRNA OSER1-AS1.RNA Binding Protein Immunoprecipitation(RIP)assays were performed to validate binding of ELAVL1 and AGO2 to lncRNA OSER1-AS1.Dual-luciferase reporter assays were performed to verify the binding effects between lncRNA OSER1-AS1 and miR-17-5p,and between lncRNA OSER1-AS1 and ELAVL1.The qRT-PCR were used to detect the relationship between lncRNA OSER1-AS1 and miR-17-5p.The WB were used to detect the protein expression levels of ELAVL1 in nucleus and cytoplasm of NSCLC cells after knockdown or overexpression of lncRNA OSER1-AS1.The WB were used to detect the protein expression levels of MYC and BCL2L11 after knockdown or overexpression of lncRNA OSER1-AS1 in NSCLC cells.Results:1.The starBase website showed that there was a potential binding site of miR-17-5p in lncRNA OSER1-AS1.RIP assays confirmed that AGO2 protein can bind to lncRNA OSER1-AS1.Dual-luciferase reporter assays confirmed that mi R-17-5p can target lncRNA OSER1-AS1.2.Overexpression or knockdown of miR-17-5p significantly down-regulated or up-regulated lncRNA OSER1-AS1 expression levels in NSCLC cells,respectively.On the other hand,after knockdown or overexpression of lncRNA OSER1-AS1 in NSCLC cells,miR-17-5p were up-regulatedd or down-regulated,respectively.3.Up-and down-stream in close proximity of this miR-17-5p target site,4 binding sites of RNA-binding protein ELAVL1 were supported by results from 4 independent CLIP-seq experiments in GEO database.RIP assays confirmed that ELAVL1 can bind to lncRNA OSER1-AS1.4.Dual-luciferase reporter assays confirmed that ELAVL1 can bind to lncRNA OSER1-AS1 in competition with miR-17-5p.The WB results showed that OSER1-AS1 promoted the accumulation of ELAVL1 protein in the cytoplasm of NSCLC cells.5.The results of WB indicated that knockdown of lncRNA OSER1-AS1 increased the MYC expression,whereas the overexpression of lncRNA OSER1-AS1 decreased the MYC expression.The influence of lncRNA OSER1-AS1 on the BCL2L11 expression was in the opposite direction.Conclusion: lncRNA OSER1-AS1 carried out tumor-suppressive functions by acting as ELAVL1 and miR-17-5p decoy to keep them away from target m RNAs. |
PDF Full Text Request