98% at 24, 48, 72 and 96 h, learn more respectively (P < 0.05) compared with control group at each time point. We observed the similar results AZD3965 in Siha cells with viabilities of 90.45%, 84.16%, 71.09% and 60.47% at 24, 48, 72 and 96 h after transfection, respectively (P < 0.05) compared with control group at each time point. Figure 3 Viability of Hela and Siha cells at different time after transfection determined by MTT assay. Viabilities of Hela and

Siha cells in transfection group were 91.47%, 86.74%, 78.92%, 48.98% and 90.45%, 84.16%, 71.09%, 60.47% at 24, 48, 72 and 96 h, respectively. (n = 3, *P < 0.05, **P < 0.01, compared with control group). Effects of DNMT1 silencing on gene demethylation and mRNA expression level in Hela cell Methylation status and mRNA expression level of seven repressive genes in Hela cells were performed with MeDIP-qPCR assay and Real-time PCR (Figure 4) compared with drug group(5-aza-dC, methylase inhibitors), control group and blank group. Specifically, PAX1, SFRP4 and TSLC1 possessed higher levels of methylation, while CHFR and FHIT were relatively lower. Except for FHIT and PTEN, the rest five suppressor

genes CCNA1, CHFR, PAX1, SFRP4 and TSLC1 in transfection group displayed lower level of methylation status compared with control group (P < 0.01), which decreased to 34.42%, 15.57%, 22.36%, 52.09% and 35.53%, respectively. The effects of DNMT1-siRNA and 5-aza-dC treatment were performed the identical phenomenon. The relative mRNA levels of seven repressive genes

were detected by Real-time PCR. It’s clear that the expression of PTEN was higher than other genes. Except for BVD-523 FHIT and PTEN, the expression levels of CCNA1, CHFR, PAX1, SFRP4 and TSLC1 in transfection group were higher than those in control group, with relative mRNA levels increased 6.13, 10.39, 4.98, 4.87 and 3.51 folds, respectively. Figure 4 Effects of DNMT1 silencing on gene methylation and mRNA expression of seven tumor suppressor Phosphoprotein phosphatase genes in Hela cells assayed by MeDIP combined with Real-Time PCR. Except for FHIT and PTEN, the rest five suppressor genes CCNA1, CHFR, PAX1, SFRP4 and TSLC1 in transfected group displayed lower level of methylation with increased mRNA expression when compared with control group. (n = 3, **P < 0.01). Effects of DNMT1 silencing on gene demethylation and mRNA expression level in Siha cell Figure 5 showed the methylation status and mRNA levels in Siha cells were similar to those in Hell cells. PAX1, SFRP4 and TSLC1 possessed higher level of methylation status, while PTEN and FHIT were relatively lower. Except for FHIT and CHFR, the rest five repressor genes CCNA1, PAX1, PTEN, SFRP4 and TSLC1 in transfection group displayed lower level of methylation compared with control group (P < 0.01), which decreased to 35.21%, 23.75%, 19.51%, 33.15% and 38.04%, respectively. Furthermore, the relative mRNA expression level of PTEN was higher than other genes.