Role Of DNA Methyltransferase 1 In DNA Methylation Of Pancreatic Cancer

The incidence rate of pancreatic cancer has an ascending trend in recent years. There were approximately 27000 new cases of pancreatic cancer,which resulted in around 26000 deaths each year in the United State and that pancreatic cancer currently ranked as the fourth leading cause of cancer death in the United States and the tenth in China.Pancreatic cancer are rarely curable and the prognosis of patients is poor,as diagnosis usually occurs at a time when the tumor has metastasis to adjacent organs and surgical removal of the tumor has become impractical.The 5-year survival rate is dismal. Despite the high mortality rate associated with pancreatic cancer,the causes of pancreatic cancer have not been identified which prevent the development of the treatment.It is likely that treatment of this fatal disease will required the detailed understanding of the molecular mechanism of pancreatic cancer.Epigenetic modification is defined as a heritable,reversible change in gene expression that does not result from DNA sequence alterations.Its physiological function is regulating the cell express the proper genes in the proper time and location by histone modifications and DNA methylation thereby controlling proliferation and differentiation. Dysfunctions of epigenetic code often involve in the initiation and progression of cancer.DNA methylation is one type of epigenetic modification.Promoter hypermethylation caused gene silencing.On the contrary,promoters demethylation caused gene re-expression of tumor suppressor genes.DNA methylation is an epigenetic event in which DNA methyltransferases(DNMTs) cause the addition of a methyl group connected to the fifth carbon position of a cytosine residue in CpG dinucleotides.The mammalian DNMTs family encompasses DNMT1,DNMT2,DNMT3a and DNMT3b. DNMT1 is one of the most important factors in maintaining DNA methylation.Human cancers generally show global DNA hypomethylation accompanied by region-specific hypermethylationRecently,many studies have identified that the abnormal activation of DNMTs is associated with many common human malignancies including pancreatic cancer.The strong relationship between DNMTs and pancreatic cancer suggests that it may be a new target for anticancer therapy.Specific targeting of DNMTs resulted in gene re-expression of tumor suppressor genes.DNA methylation of multiple tumor-related genes in association with over-expression of DNMT1 protein.Over-expression of DNMT1 plays an important role during multistage carcinogenesis of the pancreas from early precancerous stages to malignant progression.Selective depletion of DNMT1 reduced gene-specific demethylation and re-expression of tumor-suppressor genes in human non-small cell lung cancer and breast cancer cells.Genetic disruption of both DNMT1 and DNMT3b reduced genomic DNA methylation by greater than 95%and suppressed the growth of colorectal cancer cells.In theory,depletion of DNMT1 activity can reduce methylation and causes re-expression of tumor-suppressor genes.It may be the ideal target for therapeutic strategy against pancreatic cancer.Recent years,RNA interference(RNAi) technology shows up its superiority in silencing the expression of target gene efficiently and has a huge potential application value.By far,RNAi-based tumor target gene therapy has been rapidly developed and mighty progress has been made.This study focus on DNMT1 gene.At first,we detect the expression of DNMT1 in pancreatic cancer and paracancerous tissues and investigate the relationship between the level of DNMT1 expression and clinicopathological significance.Secondly,The changes of proliferation,apoptosis and cell cycle after DNMT1 silencing in pancreatic cancer cell were also evaluated.Finally,we detect the activity of DNMT1 and the corresponding changes of DNA methylation.It is investigated the antitumor of molecular mechanism of DNMT1 siRNA in pancreatic cancer.The whole study includes five parts.1.The expression of DNMTs mRNA in pancreatic cancer tissue.Objective To detect the expression of DNMTs mRNA in normal pancreas tissues, pancreatic cancer and paracancerous tissues and investigate the effects of DNMTs in pancreatic cancer.Methods and materials 22 samples of pancreatic cancer tissues,paired paracancerous tissues and 5 samples of normal pancreas tissues were collected.The levels of DNMT1, DNMT3a and DNMT3b mRNA were detected by Real-time RT-PCR,then the expression of DNMT1,DNMT3a and DNMT3b mRNA were analyzed.Results Compared with normal pancreas tissues,the values of relative quantification(RQ) of DNMT1,DNMT3a and DNMT3b mRNA in human pancreatic cancer tissues were significant higher than those in paracancerous tissues(2.32(1.17-5.17) vs 0.78(0.07-3.14), P=0.024;9.41(0.38-41.95) vs 2.00(0.22-24.88),P=0.022;7.78(0.36-43.78) vs 4.23(0.12-18.51),P=0.025,respectively).Conclusions The levels of DNMT1,DNMT3a and DNMT3b mRNA were higher in human pancreatic cancer tissues than in paracancerous tissues.DNMTs might play an important role in carcinogenesis of pancreatic cancer.2.Expression of DNMT1 protein in pancreatic cancer tissues and its Clinicopathological significance.Objective To detect the levels of DNMT1 protein in pancreatic cancer and paracancerous tissues.The relationship between levels of DNMT1 protein and clinicopathological significance in patients with pancreatic cancer was then investigated.Methods and materials Pancreatic cancer tissue samples and paired paracancerous tissue samples were obtained from 30 pancreatic cancer patients who underwent curative pancreectomy at Changhai Hospital,from Jan.2006 to Mar.2008.Clinical data were collected.In all patients,the pancreatic ductal adenocarcinoma were confirmed histologically,based mainly on the examination of sections stained with hematoxylin and eosin.Expression of DNMT1 was detected by streptavidin peroxidase immunohistochemistry techniques.The positive cells were nuclei staining.For each sample,at least 500 cells were randomly counted.If the total cells in one section were less than 500 cells,all the cells were counted.The incidence of DNMT1 immunoreactivity in each sample was expressed as a percentage.If no tumor cells were stained,the result was judged as negative.The relationships between expression of DNMT1 and clinicopathological findings were then analyzed.Results Positive cells were color brown.Specific DNMT1 staining was seen in the nuclei of the cancer cells,while it was rarely seen in non-cancerous lesions.The index of expression of DNMT1 in human pancreatic cancer tissues and paracancerous tissues were 54.5%±21.2%and 10.9%±15.0%(P＜0.05).The high DNMT1 group(n=19) with 54.5% or more of the cancer cells stained DNMT1 positively and the low DNMT1 group(n=11) with less than 54.5%cancer cells stained DNMT1 positively.The high expression of DNMT1 correlated significantly with a more advanced clinical stage(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 clinicopathological variables including age,gender,tumor location,tumor size,tumor differenciation,the concentration of CEA and CA199 in serum could be found.Conclusions DNMT1 was highly expressed in pancreatic cancer tissues.High expression of DNMT1 might have an important role in the aggressiveness,lymph node metastasis and neural invasion.3.The changes of DNMT1 siRNA on methylation of pancreatic cancer cells.Objective To evaluate the role of transient transfection of DNMT1 siRNA on DNMT1 activity in pancreatic cancer cell line.Elucidate the effect of DNMT1 siRNA on DNA methylation in pancreatic cancer cell.Methods and materials Transient transfection of siRNA against DNMT1 on Human pancreatic cancer cell line PaTu8988 cells were adopted by Lipofectamine.Pancreatic cancer cells were divided into five groups:Control group,Negative control siRNA (N-siRNA) group(15nM),N-siRNA group(30nM),DNMT1 siRNA group(15nM) and DNMT1 siRNA group(30nM).PaTu8988 cells were plated 24h prior to transfection then were transiently transfected DNMT1 siRNA or N-siRNA.At 48h after transfection,cells were collected and the total RNA and protein were extracted.Real-time RT-PCR were used to detect the level of DNMT1 mRNA of the above groups.Western blotting was performed to evaluate the expression of DNMT1 protein of the above groups to assess the efficiency of DNMT1 gene silencing.DNMTs activity was detected using DNMTs Activity Assay Kit (Colorimetric).At 48h after transfection,cells were collected and DNA were extracted then sodium bisulfite modification and bisulfite sequencing PCR(BSP) of hMLH1 and RAR-βwas performed based upon instrument.The production of BSP was purified and inserted into pMD18-T vector with ligase.The ligation mixture was used to transform E.coliDH5αwith CaCl₂,after which the cells were plated on LB plate and placed at 37℃.The next morning,the plate was examined and the white clones were picked and identified by PCR. Then the transformed E.coliDH5αwas sequenced to analyze methylation of CpG island of hMLH1 and RAR-β.Results Real-time RT-PCR analysis showed that the level of DNMT1 mRNA in DNMT1 siRNA group(15nM)(RQ=0.573±0.026) and DNMT1 siRNA group(30nM) (RQ=0.143±0.044) were significantly lower than those in Control group (RQ=1.020±0.217),N-siRNA group(15nM)(RQ=0.900±0.475),N-siRNA group(30nM) (RQ=0.938±0.327)(P＜0.01).Western blotting analysis showed that the level of DNMT1 protein of DNMT1 siRNA(15nM) group and DNMT1 siRNA(30nM) group were lower than that of other groups.DNMT activity in DNMT1 siRNA(15nM) group and DNMT1 siRNA(30nM) group were significantly inhibited than those in other groups(P＜0.05). Methylated CpG sites of hMLH1 and RAR-βin DNMT1 siRNA group were decreased than those in Control group(P＜0.05).Conclusions Transfection of DNMT1 siRNA effectively inhibits the expression of DNMT1 mRNA and protein in PaTu8988 cell.Transfection of DNMT1 siRNA effectively inhibited the DNMT1 activity and methylated CpG sites in promoter of hMLH1 and RAR-βin human pancreatic cancer cell lines were also decreased by DNMT1 siRNA.4.Effects of siRNA against DNMT1 on pancreatic cancer cell proliferation and cell cycle.Objective To evaluate the effects of transient transfection of siRNA against DNMT1 on proliferation and cell cycle of pancreatic cancer cell.Methods and materials Transient transfection of siRNA against DNMT1 on Human pancreatic cancer cell line PaTu8988 cells were adopted by Lipofectamine.Cell proliferation of the above groups was analyzed by WST-8 assay 48h after transfection. Flow cytometry was performed to analyze cell cycle.Real-time RT-PCR was used to detect the change of expression of cyclin-dependent kinase inhibitor 1A(CDKN1A) mRNA after DNMT 1 siRNA interference.Results WST-8 assay showed that at 48h after infection,Cell survival rate of DNMT1 siRNA group(30nM) was declined to 63.1%±14.2%from control group(100%±12.0%), N-siRNA group(15nM)(86.3%±8.2%),N-siRNA group(30nM)(76.6%±7.3%) and DNMT1 siRNA group(15nM)(82.9%±5.8%)(P＜0.01);Flow cytometry analysis showed there were strong G2/M cell cycle arrest in DNMT1 siRNA group(15nM) and DNMT1 siRNA group(30nM).The percentage of G2/M phase cells was significantly lower in cells infected with DNMT1 siRNA than those in other groups(P＜0.05).The level of CDKN1A mRNA in DNMT1 siRNA group(30nM)(RQ=54.918±5.774) were significantly higher than those in Control group(RQ=1.050±0.363),N-siRNA group(15nM) (RQ=5.185±0.165),N-siRNA group(30nM)(RQ=7.352±0.074) and DNMT1 siRNA group(15nM)(RQ=5.761±0.271)(P＜0.05).Conclusions Transfection of DNMT1 siRNA induces G2/M cell cycle arrest.Transfection of DNMT1 siRNA suppresses the cell proliferation of pancreatic cancer cell.In addition, transfection of DNMT1 siRNA increased the expression of CDKN 1A mRNA.5.Effects of siRNA against DNMT1 on pancreatic cancer cell apoptosis.Objective To evaluate the effects of transient transfection of siRNA against DNMT1 on apoptosis of pancreatic cancer cell and the expression of Bcl-2 and Bax mRNA.To investigated the apoptosis of molecular mechanism of DNMT1 siRNA in pancreatic cancer.Methods and materials Transient transfection of siRNA against DNMT1 on Human pancreatic cancer cell line PaTu8988 cells were adopted by Lipofectamine.Flow cytometry was performed to analyze apoptosis.Real-time RT-PCR was used to detect the change of expression of antiapoptotic(Bcl-2) and proapoptotic(Bax) genes mRNA after DNMT 1 siRNA interference.Results Flow cytometry analysis showed that cell apoptosis rate was increased from Control group(7.51%±1.12%),N-siRNA group(15nM)(9.45%±0.98%),N-siRNA group (30nM)(8.84%±1.44%),DNMT1 siRNA group(15nM)(10.01%±0.66%) to DNMT1 siRNA group(30nM)(14.94%±2.89%)(P＜0.05),at 48h after transfection of DNMT1 siRNA.The level of Bcl-2 mRNA were no different in Control group(RQ=1.004±0.108), N-siRNA group(15nM)(RQ=0.817±0.074),N-siRNA group(30nM)(RQ=0.672±0.016), DNMT1 siRNA group(15nM)(0.522±0.050) and DNMT1 siRNA group(30nM) (1.143±0.386).The level of Bax mRNA in DNMT1 siRNA group(30nM) (RQ=8.000±0.579) were significantly higher than those in Control group (RQ=1.021±0.219),N-siRNA group(15nM)(RQ=1.372±0.199),N-siRNA group(30nM) (RQ=2.636±0.387) and DNMT 1 siRNA group(15nM)(RQ=1.316±0.116)(P＜0.05).Conclusions Transfection of DNMT1 siRNA induces apoptosis on pancreatic cancer cell. It may happen through up-regulated the expression of Bax mRNA.From the above results,the following conclusions can be made:1.The levels of DNMT1,DNMT3a and DNMT3b mRNA were higher in human pancreatic cancer tissues than in paracancerous tissues.2.DNMT1 was highly expressed in pancreatic cancer tissues.High DNMT1 expression might have an important role in the aggressiveness,lymph node metastasis and neural invasion.3.Transfection of DNMT1 siRNA effectively decreased the DNMT1 activity and demethylated CpG sites in promoter of hMLH1 and RAR-βin human pancreatic cancer cell.4.Transfection of DNMT1 siRNA induces G2/M cell cycle arrest,thereby suppresses the growth of pancreatic cancer cell.In addition,transfection of DNMT1 siRNA increased the expression of CDKN1A mRNA.5.Transfection of DNMT1 siRNA induces apoptosis on pancreatic cancer cell.It may happen through up-regulated the expression of Bax.In summary,DNMTs might play an important role in carcinogenesis of pancreatic cancer.The change of DNMT1 activity correlated with genes about pancreatic cancer. RNA interference against DNMT1 induces cell cycle arrest and apoptosis.DNMT1 gene may be the ideal target for therapeutic strategy against pancreatic cancer.The role of other member of DNMTs should be further studied.