Effects of DNA methyhransferase 1 gene silencing on DNA methylation of pancreatic cancer / 中华胰腺病杂志

ABSTRACT

ObjectiveTo assess the effects of DNA methyhransferase 1 ( DNMT1 ) gene silencing on DNMTs activity and methylated CpG sites of hMLH-1 in pancreatic cancer cell line PaTu8988.Methods DNMT1 siRNA and negative control siRNA was constructed by Ambion Company of United States.Then they were transfected into pancreatic cancer cell line PaTu8988 at the concentrations of 15,30 nmol/L,and the cells without transfection was used as the control group.Real-time PCR and Western blotting were applied to detect the DNMT1 mRNA and protein expression,and DNMTs activity was detected by using DNMTs activity assay kit.Change of methylation of CpG island of hMLH-1 was detected by bisulfite sequencing PCR (BSP).The expression of hMLH-1 mRNA was detected by Real-time PCR.ResultsAt 48 h after transfection,Realtime RT-PCR analysis showed that the levels of DNMT1 mRNA in DNMT1 siRNA group ( 15 nmol/L) and DNMT 1 siRNA group (30 nmol/L) were 0.573 ± 0.026 and 0.143 ± 0.044,which were significantly lower than those in control group 1.020 ±0.217 and negative siRNA 15 nmol/L group 0.900 ±0.475,and negative siRNA 30 nmol/L group 0.938 ± 0.327 (P ＜0.05 ).Western blotting analysis showed that the level of DNMT1 protein of DNMT1 siRNA group was also lower than those of negative siRNA and control groups.DNMT activity in DNMT1 siRNA15,30 nmol/L groups was 0.364 ± 0.124and 0.250 ± 0.072,which were significantly lower than those in control group 0.931 ± 0.065and negative siRNA group 0.665 ± 0.055 and 0.472 ± 0.040.DNMT activity was positively correlated with DNMT1 mRNA expression ( r =0.69,P ＜ 0.01 ).DNMT1 RNA interference decreased 8 methylated CpG sites of hMLH-1 to 1 site.Concluslons DNMT1siRNA can specifically inhibit the expression of DNMT1 gene of PaTu8988 and DNMT activity,and can decrease methylated CpG sites of hMLH-1 gene.