| Background?Objective:Cervical cancer is one of the most common gynecological malignancies.In recent years,the occurance of cervical cancer have showed a significantly younger tendency,and it has seriously endangered the health and lives of women.High-risk HPV infection is the main risk factor for cervical cancer,of which HPV16 and 18 are the most important.Long non-coding RNA(IncRNA)is a type of non-coding RNA with a length of 200-100000 nucleotides,which have complex and diverse functions.LncRNA can regulate genes at the phenotype,transcriptional and post transcriptional levels,it also plays an important role in the development of tumors.LncRNA HOTAIR was the first IncRNA which was found to have a trans regulatory function and it was highly expressed in cervical cancer.This study mainly focus on the function of HOTAIR acting as a competitive endogenous RNA via miR-214 in HPV16 positive cervical cancer cells to regulate cell proliferation and apoptosis and its mechanisms.Methods:1.Cultivation of HPV negative C-33A cells,HPV16 positive SiHa cells and immortalized cervical epithelial Endl/E6E7 cells which was transformed by HPV 16 E6 and E7 oncogene.Real-time fluorescence quantitative PCR was used to detect the expression levels of HPV16E7,HOTAIR,and miR-214 in these cells.2.To investigate the effects of HOTAIR and miR-214 on proliferation and apoptosis of these three cell lines,small interference RNA of HOTAIR,miR-214 mimics and their negative controls were transfected into C-33A,Endl/E6E7,and SiHa cells,CCK8 and EdU incorporation assay were used to detect the changes in cell proliferation,and the apoptosis level of cells were detected by flow cytometry.3.Using starbase v2.0 to predict HOTAIR-miR-214 binding sites,dual luciferase reporter system assay was used to verify the binding sites.4.After knocking down the expression of HOTAIR in C-33A,Endl/E6E7 and SiHa cells and overexpressing HOTAIR in C-33A cells,real-time fluorescence quantitative PCR was used to detect the expression levels of miR-214.The expression levels of HOTAIR and miR-214 were detected after HPV16E7 knockdown and overexpression in these three cell lines.5.The target genes of miR-214 were predicted by TargetScan7.2,PicTar and MirTarget2,and the intersection of these three results was taken as candidate target genes,and the enrichment function and pathway analysis of the candidate target genes were performed by DAVID database.The expression of ?-catenin mRNA and protein was detected after knocking down of HOTAIR and overexpressing of miR-214 in these three cells and HOTAIR overexpressed in C-33A cells.Results:1.Real-time fluorescence quantitative PCR results showed that,compared with HPV negative cells C-33A,HPV16E7 and HOTAIR expression levels were significantly higher in Endl/E6E7 and SiHa cells(P<0.01),while the expression level of miR-214 was significantly lower(P<0.001).2.The results of CCK8 and EdU assay and apoptotic assay showed that after knocking down of HOTAIR and overexpressing of miR-214 in Endl/E6E7,SiHa cells,the proliferate abilities of these cells were significantly inhibited(P<0.05).meanwhile the apoptotic level of these cells increased significantly(P<0.05),however there were no significant changes in C-33A;however,when HOTAIR was overexpressed in C-33A cells,cell proliferation was significantly upregulated(P<0.01),and the level of apoptosis was significantly decreased(P<0.05).3.Starbase v2.0 predicts that the binding site of HOTAIR and miR-214 is located at the position of 1794-1814 bp on HOTAIR variant 1,we adopted dual luciferase reporter system experiment to verify the binding site,after transfection of miR-214 mimics,the fluorescence signal showed a significant decrease(P<0.01),the binding sites of miR-214 on HOTAIR was comfirmed.4.After knocking down of HOTAIR expression in Endl/E6E7 and SiHa cells,the expression of miR-214 was significantly upregulated(P<0.001),but there was no significant change in C-33A cells(P>0.05),while after overexpression of HOTAIR in C-33A,the expression of miR-214 was significantly downregulated(P<0.05);when HPV16E7 knockdown in these three cells,the expression of HOTAIR in Endl/E6E7 and SiHa cells was significantly downregulated(P<0.05),at the same time,the expression of miR-214 was significantly upregulated(P<0.05),but there was no significant change in C-33A cells(P>0.05),and when HPV16E7 was overexpressed in C-33A,Endl/E6E7,and SiHa cells,the expression level of HOTAIR in these three cells increased significantly(P<0.05),while the expression of miR-214 significantly decreased(P<0.05).5.Through target gene prediction of miR-214 and bioinformatics analysis,we found that miR-214 target gene was significantly enriched in the Wnt signaling pathway(P<0.001),and ?-catenin was a validated target of miR-214.After knocking down HOTAIR and overexpressing miR-214 in Endl/E6E7 and SiHa cells,the mRNA and protein levels of ?-catenin were both significantly downregulated(P<0.05).And after overexpression of HOTAIR in C-33A cells,the mRNA and protein levels of ?-catenin were significantly upregulated(P<0.05).Conclusion:HOTAIR is highly expressed in HPV16 positive cervical cancer cells.HOTAIR can act as a competitive endogenous RNA and induces the expression of?-catenin through competing binding to miR-214,which promotes the occurance and development of HPV16 positive cervical cancer.HOTAIR could be a potential therapeutic target for HPV16 positive cervical cancer and it could also be used as an indicator of prognosis in cervical cancer. |
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