The Inhibition Effect Of 5-Aza-dC And Radiotherapy On Cervical Cancer Cell Lines Proliferation And The Correlation With P16 And MGMT Gene Expression

BackgroundCervical cancer is one of the most common female genital malignant tumors,with high incidence and high mortality.The age of onset tends to be younger,and the choice of its treatment is not much.Therefore,it is critical to investigate the molecular mechanisms involved in the development and progression of cervical cancer.p16(also named cyclin-dependent kinase inhibitor 2A,CDKN2A)and O-6-methylguanine-DNA methyltransferase(MGMT)are important tumour suppressor genes.Recent studies have reported that these two genes are highly methylated in cervical cancer.But available data show that the levels of p16 and MGMT protein expression are not related to their promoter methylation status and are increased in cervical cancer.Thus,it is important to elucidate the relationship between p16 and MGMT methylation and expression as well as their roles in cervical cancer.5-aza-2'-deoxycytidine(5-Aza-dC)is a DNA methyltransferase inhibitor that restores expression of some tumour suppressors(which are highly methylated in certain tumours)to combat cancer progression.Although this compound has been clinically used to treat leukaemia,its therapeutic effects on other cancers and its specific mechanisms remain poorly characterized.The present study aimed to determine the relationship among p16,MGMT and 5-Aza-dC and to identify potential therapeutic targets and treatment modalities for cervical cancer.Objectives:To investigate the inhibition effect of 5-Aza-dC and radiotherapy on cervical cancer cell line proliferation and the correlation with p16 and MGMT gene expression.Methods:Four cervical cancer cell lines(HeLa,SiHa,C33A and CaSki)were treated with 5-Aza-dC,and the methylation specific PCR(MSP)was used to detect the methylation of p16 and MGMT.Cervical cancer cell lines were co-transfected with p16 or/and MGMT siRNAs,p16 or MGMT over expression vector,overexpression vector of human papillomavirus(HPV)E6 or E7 gene,and then treated with 5-Aza-dC or radiotherapy.The expression of p16 and MGMT was detected by fluorescence quantitative PCR and Western blot.MTT was used to detect cell proliferation,and Annexin V-FITC/PI double staining or PI was used to detect cell apoptosis and cell cycle.The expression of E6,E7,p53,p21 and Rb was further detected by fluorescence quantitative PCR.Results:Cervical cancer cell lines were stimulated with 5-Aza-dC and the results found that this treatment inhibited cell proliferation,and induced apoptosis;additionally,methylation of p16 and MGMT was reversed,though their expression was suppressed.5-Aza-dC inhibited HPV E6 and E7 expression,and up-regulated p53,p21 and Rb expression.Cells transfected with siRNAs targeting p16 and MGMT as well as cells stimulated with 5-Aza-dC were arrested in S phase,and the expression of p53,p21 and Rb was up-regulated more significantly.However,when cells were stimulated with 5-Aza-dC after transfection with siRNAs targeting p16 and MGMT,proliferation decreased significantly,and the percentage of cells in the sub-G1 peak and in S phase was significantly increased,suggesting a marked increase in apoptosis.Furthermore,radiotherapy caused cells to arrest in G2/M phase,but cells transfected with p16-and MGMT-targeted siRNAs followed by radiotherapy exhibited a significant decrease in proliferation and were shifted toward the sub-G1 peak,also indicating enhanced apoptosis.In addition,the effects of 5-Aza-dC and radiotherapy were most pronounced when MGMT expression was down-regulated.Conclusions:1.5-Aza-dC reduced the promoter methylation of p16 and MGMT while inhibiting their expression in cervical cancer cell lines;2.5-Aza-dC inhibits the proliferation of cervical cancer cell lines and promotes their apoptosis,causes cells to arrest in S phase;down-regulation of p16 or MGMT can enhance the anti-tumor effect of 5-Aza-dC;3.Radiotherapy caused cervical cancer cell lines to arrest in G2/M phase,and down-regulation of p16 or MGMT can increase the sensitivity of cells to radiotherapy;4.5-Aza-dC may promote the expression of p53,p21 and Rb by inhibiting the expression of HPV E6 and E7 genes.P16 and MGMT may be involved in the process of gene expression and regulation,and the specific mechanisms need to be further explored.Novelty:1,The interaction between the down-regulation of p16 and MGMT,5-Aza-dC and radiotherapy was investigated;2.The study of the regulatory mechanism of p16,MGMT and 5-Aza-dC on the growth of cervical cancer cells can provide new therapeutic targets and new therapeutic ideas for cervical cancer.