A Preliminary Research On The Function Of NCAPH And Its Mechanism In The Carcinogenesis Of Cervical Cancer

Background:Cervical cancer is one of the most common gynecological tumors in the world,and human papillomavirus(HPV)infection is its causative agent.HPV is a kind of double-stranded DNA virus,and HPV E6 and E7 are the key oncoproteins for the malignant transformation of cervical cells.However,HPV infection is only the first hit in the transformation of the host cells,and HPV infection alone can not induce the carcinogenesis of cervical cancer.Once HPV infects the cervical epithelium,the changes of the molecular signaling pathways it induced lead to the malignant transformation of the host cells.However,the detailed molecular mechanism in the development of cervical cancer remains to be further elucidated.Non-SMC Condensin I Complex Subunit H(NCAPH)is mainly involved in the transformation of interphase nuclear chromatin into a highly helical nuclear chromosome.Previous studies have shown that NCAPH is associated with poor prognosis in non-small cell lung cancer,suggesting that NCAPH may be involved in the development of human malignancies,however,the detailed mechanism is largely unknown.Aim:To assess the expression level of NCAPH in cervical cancer tissue.To demonstrate the effects of NCAPH on the growth of cervical cancer cells and its possible mechanism.To examine the regulatory relationship between HPV E7 and NCAPH in cervical cancer.Methods:1)Total RNAs were extracted from cervical tissue and cervical cancer cell lines and reverse-transcribed into cDNAs.QPCR method was used to detect the mRNA expression of NCAPH,and immunohistochemical method was used to detect the protein level of NCAPH.The difference between normal cervical tissue and cervical cancer tissue was analyzed.2)Cervical cancer cell lines HeLa and SiHa were cultured routinely and transfected with double-stranded small interfering RNA(siRNA)to knock down the expression of NCAPH.Cell proliferation was detected by cell viability assay(CCK-8 kit),and colony formation assay.The ability of cell migration and invasion was examined by transwell migration and invasion assay.3)Western Blot and real-time quantitative PCR method were used to detect the expression of related proteins after siRNA transfection.4)JASPAR and PROMO software were used to predict the transcription factor that binds to the NCAPH gene promoter region,and the transcription factor E2F1 with the highest score was selected.The mRNA and protein expression of NCAPH were examined after knockdown or over-expression of the transcription factor E2F1 in HeLa and SiHa cells;5)The vectors containing the sequences of NCAPH promoter region and the E2F1 over-expression plasmid were constructed and then tranfected into 293T cell.Luciferase reporter gene assay was used to determine whether the transcrption factor E2F1 could bind to the promoter region of NCAPH and initiate its transcription.6)RPE1 E7 and its controlled cell line RPE1 were cultured routinely,and the expression of E7,NCAPH and transcription factor E2F1 was examined.Hela and SiHa cells were treated with E7 siRNA,the expression of NCAPH and transcription factor E2F1 was further examined.RPE1 E7 cells were treated with E2F1 siRNA,and the expression of NCAPH was further examined.Results:1)NCAPH expression can be detected in three cervical cancer cell lines(HeLa,SiHa and Caski).Compared with normal cervix,NCAPH expression level was significantly increased in cervical cancer tissue;2)After NCAPH siRNA transfection,cell proliferation and colony formation ability of HeLa and SiHa cells were decreased significantly.3)After interference of the expression of NCAPH in cervical cancer cells,the migration and invasion ability of the cells was significantly inhibited,In the meanwhile,the expression level of epithelial-related protein ZO-1 was significantly increased,while the expression level of mesenchyma-related protein Vimentin and Snail was significantly decreased.4)After interference of the expression of NCAPH in cervical cancer cells,the total AKT protein did not change significantly,while the phosphorylation of AKT(Ser473)protein expression was reduced significantly;5)The transcription factor E2F1 can upregulate NCAPH transcription by binding to the promoter region of NCAPH,and HPV E7 might upregulate NCAPH expression via the transcription factor E2F1.Conclusions:NCAPH may be a novel oncogene in the development of cervical cancer.It can promote cell proliferation of cervical cancer cells and enhance its ability of invasion and metastasis by promoting EMT.HPV E7 might upregulate NCAPH expression via the transcription factor E2F1 in the transcription level.