Study Of The Mechanism Of Action Of Centromere Protein F(CENPF) On Cervical Cancer Cells

Background Cervical cancer is one of the deadliest types of cancer that poses a serious threat to women’s health.Centromere protein F(CENPF)is a key protein in mitosis and regulates a variety of cellular processes,including chromosome separation during mitosis.Bioinformatic analysis has shown that CENPF is overexpressed in cervical cancer,but its expression level and exact biological function and mechanism of action as a major regulator remain unclear in cervical cancer.Objective The aim of this study was to investigate the expression levels of CENPF in human cervical cancer tissues and cell lines through in vivo and in vitro experiments,and to explore the influence of the change of CENPF expression level on the biological function of cervical cancer cells,as well as the possible molecular mechanisms regulating this process.Methods1.The Expression of centromere protein F in human cervical cancer tissues.The mRNA levels of CENPF in existing cervical cancer and normal cervical tissues were compared in the TCGA database.Immunohistochemical methods were used to analyse the expression of CENPF in human cervical cancer tissues and normal cervical tissues.2.The relevance of centromere protein F to biological functions in cervical cancer cells Western-Blot assay was used to detect the expression of CENPF in cervical cancer cell lines(HeLa,SiHa,CaSki,HT-3)and normal cervical epithelial cells(Hcer Epic).Cell lines with stable knockdown of CENPF were constructed by lentiviral transfection of cervical cancer cells(Hela and Siha),and the effect of CENPF on the biological function of cervical cancer cells was investigated by Western-Blot assay,Cell Counting Kit-8 cytotoxicity Western-Blot assay,cell cloning assay,flow cytometry,cell scratching assay,cell Transwell assay and transmission electron microscopy assay were used to investigate the effect of CENPF on the biological functions of cervical cancer cells.3.Centromere protein F knockdown induced ferroptosis in cervical cancer cells via inhibition of the Nrf2/HO-1 pathway The expression of ferroptosis-related proteins-glutathione peroxidase 4,solute carrier family 7 member 11,Nrf2,HO-,p53 and metal transporter 1(GPX4,XCT,Nrf2,HO-1,p53 and DMT1 proteins)in cervical cancer cell lines(Hela and Siha)after knockdown of CENPF was measured by western-blot assay,and the expression of ferroptosis-related proteins was measured by Cell Counting Kit-8 cytotoxicity assay,malondialdehyde(MDA)assay,reactive oxygen species(ROS)assay and western-blot assay after addition of Nrf2 activator(tertiary butylhydroquinone,TBHQ).In an in vivo xenograft model,tumour cells were inoculated subcutaneously in nude mice and treated with saline or TBHQ at the same time,tumour volumes were measured at intervals,and after 21 days of incubation,mice were dissected by execution,tumour weights were measured and dissected tumours were assayed for CENPF protein,ferroptosis-related proteins(GPX4,XCT,Nrf2,HO-1,p53 and DMT1)using immunohistochemical staining and Western-Blot.Nrf2,HO-1,p53 and DMT1 proteins)in tumour tissues of mice,and further validate the mechanism of CENPF-induced ferroptosis in cervical cancer cells by in vivo experiments.Results1.Centromere protein F is overexpressed in human cervical cancer tissues The mRNA levels of CENPF in existing cervical cancer and normal cervical tissues were compared in the TCGA database,and significantly high levels of CENPF mRNA were observed in cervical cancer tissues.Immunohistochemical staining analysis revealed that CENPF was significantly overexpressed in human cervical cancer tissues and that CENPF may function as an oncogene in cervical cacncer.2.Knockdown of centromere protein F inhibits the biological effects of cervical cancer cells and triggers ferroptosis in cervical cancer cells In vitro experiments showed that CENPF was highly expressed in cervical cancer cell lines(HeLa,SiHa,CaSki,HT-3)and that knockdown of CENPF in cervical cancer cell lines(Hela and Siha)significantly inhibited biological effects such as cell colony formation,cell proliferation,cell migration and cell invasion,and significantly blocked the G2 phase of the cell cycle,while inducing ferroptosis.3.Centromere protein F knockdown induced ferroptosis in cervical cancer cells via inhibition of the Nrf2/HO-1 pathway In cervical cancer cell lines,knockdown of CENPF inhibited GPX4,xCT,Nrf2,HO-1 protein expression and promoted p53 and DMT1 protein expression.Increased production of reactive oxygen species and lipid peroxidation was detected in cervical cancer cell lines knocked down with CENPF.In cell lines treated with added Nrf2activator(TBHQ),TBHQ significantly inhibited reactive oxygen species production and lipid peroxidation in CENPF knockdown cells,and ferroptosis-associated proteins GPX4,XCT,Nrf2 and HO-1 were up-regulated,while DMT1 and p53 were down-regulated.In vivo experiments showed that knockdown of CENPF inhibited tumour growth in nude mice in vivo and was promoted by TBHQ treatment,with GPX4,XCT,Nrf2 and HO-1 up-regulated and DMT1 and p53 down-regulated in TBHQ-treated mice.Conclusion Significantly high expression of centromere protein F in cervical cancer patients affects the biological functions of cervical cancer cells.Knockdown of centromere protein F induced ferroptosis in cervical cancer cells,which may be achieved by inhibiting Nrf2/HO-1 pathway.Therefore,centromere protein F could be used as a prognostic biomarker and a new potential therapeutic target for cervical cancer patients.