| Pancreatic carcinoma is one of the most malignant cancer in digestive system andthe malignant is very high.Is usually been diagnosed when the advanced diseaseaccure. The relationship between smoking and pancreatic cancer mainly concentratedin the level of epidemiological investigation and retrospective study have beenstudied by the current domestic and foreign scholars, but the mechanism of pancreaticcancer caused by smoking has been studied less. As everyone know,there are manyrisk factors of pancreatic cancer, long time smoking is one of them. Nicotine--as themain material of smoke, is closely related with the occurrence and development ofpancreatic cancer. DNMTs has the ability to catalytic DNA methylation from thebeginning, use the non-methylated DNA as template,catalytic new methylation sites,in many tumor cells show high expression of DNA methylation,this can be regardedas a characteristic change of tumor cells.In the pre-experiments we observed the proliferation and apoptosis after impactof nicotine on human pancreatic ductal epithelial cells in vitro with differentconcentrations and different times. Result display:nicotine in the100μmol/L with along time can also protect the cell from apoptosis which may play a role in thedevelopment of pancreatic cancer. In this experiment, after100μmol/L nicotine act onthe human pancreatic ductal epithelial cells, we will detect expression and activity ofDNMT3a (DNA methyltransferase3a).Material and Methods:(1) Commonly cultured human pancreatic ductalepithelial cell (hTERT-HPNE cell);(2) After100μmol/L nicotine act on humanpancreatic ductal epithelial cells cultured in vitro with different times, use real-timepolymerase chain reaction(Real-Time PCR, RT-PCR) and Western Blot to detect theexpression of human pancreatic ductal epithelial cells DNMT3a;(3) After100μmol/L nicotine stimulation at different time,study changes of DNMT3a activity byuse DNA methyltransferase activity of DNMT3A enzyme continuous cyclic colorimetry assay kit of Genmed company;(4) The data were analyzed by softwareSPSS17.0, the measurement data were expressed as mean±standard deviation (x±s), analysis of variance (ANOVA) was used to analyze the data that accorded withnormal distribution. A p-value of less than0.05was considered statistically difference,and significant difference when the p-value was less than0.01.Results:(1) Effect of expression on DNMT3a’s mRNA after100μmol/Lnicotine act on human pancreatic ductal epithelial cells: After hTERT-HPNE cellswere treated by100umol/L nicotine for6h,12h,24h,48h,72h group and blankcontrol group, expression level of DNMT3a are obvious difference, the expression ofDNMT3a mRNA increases with100μmol/L of nicotine action time,and maximumvalue is appear at12h, then with the prolonged stimulation, expressions of DNMT3amRNA will decrease. At6h, Δ Ct compare with the control group showed significantdifference (P<0.01), but there is no specific DNMT3a amplification products insolubility curve,only has primer dimer peaks,so the DNMT3a expression of humanpancreatic ductal epithelial cells do not increase; At12h, the value of Δ Ct compareswith blank control group,6h,24h,48h and72h groups shows significant difference(P<0.01), meanwhile, there is DNMT3a specific amplification products in solubilitycurve, so when100μmol/L nicotine stimulate12h, the expression of DNMT3a mRNAis significantly higher than that in the blank control group,6h,24h,48h and72hgroups; At24h, the value of Δ Ct compares with blank control group,6h,12h,48hand72h groups shows significant difference (P<0.01), but there are DNMT3a specificamplification products and primer dimer peaks of human pancreatic ductal epithelialcells in solubility curve, it shows that the DNMT3a amplification products are existwith low expression level, so when the100μmol/L nicotine stimulate humanpancreatic ductal epithelial cells24h, the expression of DNMT3a mRNA issignificantly higher than that in the blank control group,6h,48h and72h groups, atthe same time, expression of DNMT3a mRNA is significantly lower than that in12hgroup; At48h, the value of ΔCt compares with blank control group,6h,12h,24h and72h groups shows significant difference (P<0.01), but there are DNMT3a specificamplification products and primer dimer peaks of human pancreatic ductal epithelial cells in solubility curve, so when100μmol/L nicotine stimulate human pancreaticductal epithelial cells for48h, the expression of DNMT3a mRNA is significantlyhigher than that in the blank control group,6h,and72h groups, at the same time,expression of DNMT3a mRNA is significantly lower than that in12h and24h groups;At72h, Δ Ct compare with the control group and6h,12h,24h,48h,72h groupsshowed significant difference (P<0.01), but there is only primer dimer peaks,so after100μmol/L nicotine stimulate human pancreatic ductal epithelial cells for72h, theDNMT3a expression level will not increase.(2) Effect of the expression of DNMT3aprotein when100μmol/L nicotine stimulate on pancreatic ductal epithelial cells:When each sample’s reference protein expression are not significantly different,DNMT3a protein expression has been observed when100μmol/L nicotine stimulatehuman pancreatic ductal epithelial cells for12h,and100μmol/L nicotine stimulatehuman pancreatic ductal epithelial cells for6h,24h,48h,72h and the control groupshow no obvious DNMT3a protein expression. At the same time, tumor cell group aspositive control show obvious expression of DNMT3a.(3) Effect of DNMT3Aenzyme activity when100μmol/L nicotine stimulate human pancreatic ductalepithelial cells: After hTERT-HPNE cells were stimulated by100umol/L nicotine,enzyme activity of DNMT3a will increased with the extension of time, and maximumvalue12.14*10-4unit/ug is appear at12h, then with the prolonged stimulation,enzyme activity of DNMT3a will decrease. The OD values and enzyme activitiessignificantly different between blank control group and6h,12h,24h,48h,72h, tumorcell group (p<0.01), and there is different with6h and72h group (p<0.05).Conclusion:1、100μmol/L nicotine can promote human pancreatic ductalepithelial cell DNMT3a’s mRNA expression level with the prolongation of actiontime,and maximum appear at effect time of12h,then the expression and enzymaticactivity of DNMT3a decrease with the extension of time.2、100μmol/L nicotine can promote human pancreatic ductal epithelial cellDNMT3a’s protein expression level and enzyme activity with the prolongation ofaction time,and maximum appear at effect time of12h,then the expression andenzymatic activity of DNMT3a decrease with the extension of time. 3、DNMT3a’s expression and enzymatic activity will changed when nicotineeffects on human pancreatic ductal epithelial cells, which can lead methylation ofanti-oncogene,and then lead to tumorigenesis. |
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