Study On Functions And Developmental Mechanisms Of DNMT1 In Cervical Cancer

Objective:To investigate the relationship between DNMT1 and abnormal methylation of tumor-related genes, malignant phenotype in cervical cancer, and further explore the function of DNMT1 in development of cervical carcinoma, as well as the specific regulatory mechanisms of tumor suppressor genes expression.Methods:DNMT1-siRNA was transfected into Hela, Siha cells with Lipofection method, RT-PCR, quantitative PCR(qPCR), Western blot were applied to detect DNMT1 mRNA and protein expression, evaluating gene interference effect. Cell proliferation and cell cycle, apoptosis were analyzed by MTT, Annexin V-FITC/PI double staining flow cytometry. MeDIP-qPCR, qPCR were used to measure de-methylation and mRNA re-expression of 7 tumor-related genes (CCNA, CHFR, PAX1, SFRP4, TSLC1, PTEN, FHIT) in Hela cells silenced DNMT1.Results:Relative to control, the inhibitory rates of DNMT1 mRNA levels in Hela, Siha cells were 52.54%,41.58% respectively after DNMT1-siRNA transfection 72h. The inhibitory rates of DNMT1 protein levels Hela, Siha cells were 50.31%,99.76% respectively.MTT results show that, after DNMT1-siRNA effect 24,48,72,96h, Siha cells survival rates decreased to 90.45%,84.16%,71.09%,60.47%respectively, and those of Hela cells 91.47%,86.74%,78.92%,48.98% respectively. After transfection 48h, Siha, Hela cells apoptosis rates were (19.4±2.90)%, (25.7±3.92)% respectively.The proportion of cells in S phase decreased from the control group (21.81±1.39)% to transfected group (17.54±1.84)% in Siha cells, and that of in Hela cells from the control group (29.34±1.14)% to transfected group (17.67± 1.29)%, there were significant differences (P<0.01)betweeii two groups. In Hela cells (without any treatment), all genes methylated with different degrees, enrichment of methylation of PAX1, SFRP4, TSLClwere higher than that of PTEN, FHIT. Concerning gene expression, PTEN mRNA level was higher than that of any others. After depletion DNMT1, percentage of promoter methylation decreased in five methylated genes (CCNA1, CHFR, PAX1, SFRP4, TSLC1), mRNA expression increased significantly, whereas there was no significant effect of DNMT1-siRNA on PTEN, FHIT gene. Transfection group and 5-aza-dC treatment showed the similar effect on gene demethylation and re-expression.Conclusions:1. Siha, Hela cells could reverse their malignant phenotype for DNMT1 gene depletion.2. In Hela cells, DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CCNA1, CHFR, PAX1, SFRP4, TSLC1) out of seven tumor-related genes examined.3. In Hela cells, CCNA1, CHFR, PAX1, SFRP4, TSLC1 genes presented low-expression by promoter region hypermethylation, and their methylation mainly depend on DNMT1. FHIT, PTEN genes might be present high-expression by promoter hypomethylation. There was probably no relationship between DNA methylation and DNMT1 in FHIT, PTEN gene.4. DNMT1 is an effective target for treatment of cervical cancer.