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.
ABSTRACT
Objective To investigate the expressions of DNA methyltransferase 1 (DNMT1) in pancreatic carcinoma and its clinical significance.Methods 30 samples of pancreatic cancer tissues and paired para-cancerous tissues were collected from patients who underwent curative pancreatectomy.The levels of DNMT1 mRNA were detected by real-time RT-PCR.Expressions of DNMT1 protein were detected by streptavidin peroxidase immunohistochemistry.The relationships between expression of DNMT1 and clinicopathological findings were analyzed.Results The value of relative quantification (RQ) of DNMT1 mRNA in human pancreatic cancer tissues was 2.32 (1.17 ～ 5.17 ), which was significant higher than 0.78 (0.07 ～3.14) in para-cancerous tissues(P ＜0.05).The index of expression of DNMT1 protein in human pancreatic cancer tissues was (54.5 ±21.2)% ,which was significant higher than( 10.9 ± 15.0)% in paracancerous tissues (P ＜ 0.01 ).Patients were divided into the high DNMT1 group (n = 19) with above 54.5% of the DNMT1 positively cancer cells and the low DNMT1 group ( n = 11 ) with less than 54.5% of the DNMT1 positively cancer cells.The high expression of DNMT1 was correlated significantly with clinical staging ( x2 =6.897, P = 0.029), lymph node metastasis ( x2 = 4.739, P = 0.029) and neural invasion ( x2 = 5.44, P =0.020).On the other hand, no association between DNMT1 expression and age, gender, tumor location,tumor size, tumor differentiation, the serum CEA and CA19-9 levels could be found.Conclusions DNMT1 mRNA and protein was highly expressed in pancreatic cancer tissues.High expression of DNMT1 might be related to the aggressiveness of pancreatic cancer, lymph node metastasis and neural invasion.