Detection Of The Methylation Status Of The ELA3B And NPTX2 Genes In Pancreatic Cancer And The Function Studies On NPTX2 Gene

Pancreatic carcinoma is a malignancy of the pancreas.The prognosis of patients with pancreatic cancer is extremely poor,with overall 5-year survival rate of approximately 5%. The incidence rate of pancreatic cancers has been growing in recent years.It was reported that there were approximately 200 thousand new cases of pancreatic cancer which accounted for 2%of total malignancies.The incidence of pancreatic cancers was near to mortality.In China,the incidence of pancreatic cancers increased year by year with the improvement of living standard of the people and the changes of the structure of food and drink.Statistical results in Shanghai indicated that the morbidity rates of male and female were 9.6 per ten thousand and 9.2 per ten thousand in 1995,which increased 7 to 8 times. However,very little is known about the exact molecular mechanism in the formation of pancreatic carcinoma.Epigenetic refers to the study of mechanisms that control somatically heritable gene expression states without changes in the underlying DNA sequence.Epigenetic mechanisms include DNA methylation,histone modifications such as acetylation and methylation,and control of mRNA expression by non-coding RNAs.DNA methylation plays a critical role in the development of mammals and the formation of neoplasm,and is potentially useful to the diagnosis and therapy of neoplasm.In this study,methylation specific polymerase chain reaction(MSP) and other techniques were used to study the molecular mechanism of down-expression of ELA3B and NPTX2 genes in pancreatic cancers,and to investigate the function of NPTX2 gene.1.Methylation status of ELA3B gene and the role of methylation in the regulation of ELA3B expressionObjective:To detect the expression of ELA3B gene in pancreatic tissues and to investigate the mechanism of expression regulation.Methods:To detect the ELA3B mRNA in 30 human pancreatic cancer tissues,20 human chronic pancreatitic tissues and 10 normal human pancreatic tissues by RT-PCR. Five pancreatic cancer cell lines,involving SW1990,BxPC-3,CFPAC,PANC-1 and PaTu8988,were treated by DNA methyl-transferase inhibitor 5-aza-2'-deoxycitidine (5-aza-dC).We analyzed the changes of the relative quantity of ELA3B mRNA with and without 5-aza-dC,and detected the methylation level of ELA3B promoter.Results:The positive expression rates of ELA3B mRNA in pancreatic cancer tissues, chronic pancreatitic tissues and normal pancreatic tissues were 46.7%,85.0%and 100%, respectively.ELA3B mRNA did not expressed in five detected pancreatic cancer cell lines. ELA3B mRNA was detected in SW1990,BxPC-3,PANC-1 and PaTu8988 after treatment with 5-aza-dC.However,ELA3B mRNA could not be examined in CFPAC pancreatic cancer cell line.MSP results indicated that the promoter of ELA3B gene was partially methylated in PANC-1.The promoter of ELA3B gene was hypermethylated in SW1990, BxPC-3,CFPAC and PaTu8988.Methylation index(MI) of ELA3B gene in pancreatic cancer tissues was significantly higher than those of chronic pancreatitic tissues and normal pancreatic tissues(7.02±6.31,2.33±0.97和3.31±0.75,P＜0.05).The MI of pancreatic cancer tissues with negative expression of ELA3B was significantly higher than that of pancreatic cancer tissues with positive expression of ELA3B(11.83±7.82对3.82±1.18,P＜0.05).Consults:The study firstly reported the methylation status of ELA3B gene in pancreatic cancer tissues and chronic pancreatitic tissues.It was first report that the hypermethylation of the promoter of ELA3B gene was one of the reasons that resulted in the down-regulation of ELA3B gene in pancreatic tissues.2.Expression patterns of NPTX2 gene in normal pancreas,and primary pancreatic adenocarcinomasObjective:To detect the expression of NPTX2 gene,involving the mRNA level and the protein level,in pancreatic tissues.Methods:To detect the NPTX2 mRNA in 25 human pancreatic cancer tissues and 25 adjacent normal human pancreatic tissues by real-time quantitative PCR.We analyzed the localization and the level of NPTX2 protein in human human pancreatic cancer tissues and 25 adjacent normal human pancreatic tissues by immunohistochemistry(IHC).Results:The average relative quantities(RQ) of NPTX2 mRNA in AsPC-1,BxPC-3, CFPAC,PANC-1 and PaTu8988 were 1.23±0.28,0.46±0.15,0.27±0.24,0.19±0.16 and 0.15±0.14(n=5) detected by real-time quantitative PCR.The mean RQ in 5 pancreatic cancer cells was 0.46±0.45.Quantitative RT-PCR revealed NPTX2 was expressed in both normal and tumor tissue from the same patient,but the expression levels varied greatly between tumor and the adjacent normal tissue.The mean RQ in the pancreatic cancer tissues was significantly lower than that in the paired adjacent normal tissues(0.96±0.91 vs. 2.78±51.42,P=0.000).In IHC,brain tissue was used as positive control,and NPTX2 presented brown-yellow or dark brown in neurons.NPTX2 mainly distributed in pancreatic islets of the adjacent normal tissues,while almost all the pancreatic ducts showed negative both in pancreatic cancers and the adjacent normal tissuesConsults:Comparing with the adjacent normal tissues,NPTX2 gene was down-regulated in pancreatic cancer tissues.IHC results suggested that it was negative staining in pancreatic ducts except that the pancreatic islets showed positive in adjacent normal tissues.3.Methylation analysis of NPTX2 in pancreatic cancer cell lines and pancreatic cancerous tissuesObjective:To detect the methylation status of NPTX2 gene in pancreatic cancer cell lines and pancreatic cancerous tissues.Methods:We detected the methylation status of NPTX2 gene in five pancreatic cancer cell lines and 25 human pancreatic cancer tissues and 25 adjacent normal human pancreatic tissues by real-time quantitative MSP.Five pancreatic cancer cell lines,involving AsPC-1, BxPC-3,CFPAC,PANC-1 and PaTu8988,were treated by DNA methyl-transferase inhibitor 5-aza-dC,and histone deacetylase inhibitors,such as trichostatin A(TSA) and valproic acid(VPA).We analyzed the changes of the relative quantity of NPTX2 mRNA and the methylation level of its promoter with and without treatment,and detected the protein level of NPTX2 in 5 pancreatic cancer cells with and without 5-aza-dC by immunoreactivity.Results:MSP analysis revealed the promoter region of NPTX2 gene to have an unmethylated status in the normal pancreatic tissues or a strong unmethylation with the weak methylation,while NPTX2 was frequently hypermethylated in pancreatic cancer cells. Twenty-five primary pancreatic carcinomas were also detected,and indicated completely (16.0%,4/25) and partially(84.0%,21/25) methylated.Treatment with 5-Aza-dC,TSA, VPA and 5-aza-dC/YSA could increase the expression of NPTX2 mRNA(RQ) and decrease the methylation status(MI) of NPTX2 promoter with different degree.In AsPC-1 cells,MI and RQ were 120.23 and 1.23 without any treatment,MI and RQ were 51.31 and 2.175 after 5-Aza-dC treatment,MI and RQ were 42.54 and 3.165 treated by Aza-dC and TSA simultaneously.In BxPC-3 cells,MI and RQ were 60.32 and 0.46 without any treatment,MI and RQ were 2.09 and 7.44 after 5-Aza-dC treatment,MI and RQ were 2.78 and 12.30 treated by Aza-dC and TSA simultaneously.In CFPAC cells,MI and RQ were 23.18 and 0.27 without any treatment,MI and RQ were 1.05 and 0.97 after 5-Aza-dC treatment,MI and RQ were 0.45 and 1.67 treated by Aza-dC and TSA simultaneously.In PANC-1 cells,MI and RQ were 90.07 and 0.19 without any treatment,MI and RQ were 2.11 and 6.39 after 5-Aza-dC treatment,MI and RQ were 5.59 and 2.42 treated by Aza-dC and TSA simultaneously.In PaTu8988 cells,MI and RQ were 54.49 and 0.15 without any treatment,MI and RQ were 11.04 and 0.45 after 5-Aza-dC treatment,MI and RQ were 8.86 and 0.37 treated by Aza-dC and TSA simultaneously.The results of NPTX2 immunoreactivity were consistent with the quantitative PCR analysis.Consults:The hypermethylation of NPTX2 gene promoter regulated the expression of its mRNA and protein,and was an important reason that resulted in down-regulation in pancreatic cancer.4.Initial investigation of NPTX2 gene function in pancreatic cancersObjective:To approach the roles and the mechanism of NPTX2 gene in pancreatic cancers.Methods:Full-Length cDNA for NPTX2 was amplified by PCR and subcloned into pcDNA3.1(+) vector and pGEX-4T-1,and named as pcDNA3.1(+)-NPTX2 and pGEX4T-1-NPTX2,respectively.Pancreatic cancer cell,PANC-1,was transfected by eularyotic expression vector.The transfection rate was examined by real-time quantitative PCR.The cell apoptosis was analyzed by flow cytometry.Induce the recombinant GST-NPTX2 protein,purify the objective protein,dialyze the purified protein and store the protein after freeze drying.Results:We transiently transfected a full length cDNA for NPTX2 (pcDNA3.1(+)-NPTX2) and an empty vector(pcDNA3.1(+)) into PANC-1 pancratic cancer cells,and were called trial group and control group respectively.Transfection with this construct,but not an empty vector,led to NPTX2 mRNA level significantly increasing. After transfection for 48h,the apoptosis ratios of PANC-1 cells in trial group treated by pcDNA3.1(+)-NPTX2 were significantly higher than those in control group treated by pcDNA3.1(+)(2.77±0.97 vs.1.23±0.39,P=0.005).If secreted NPTX2 was responsible for the growth inhibition,then conditioned media from the NPTX2 transfectants should also slow the growth of other pancreatic cancer cells.In order to further investigate the role of NPTX2 in pancreatic cancer,we collected the culture media after the 72h transfection,and centrifugated and added the supernatant into Patu8988 cells.This was indeed the case,as NPTX2-conditioned media significantly(P＜0.05) promoted the apoptosis of Patu8988 cells when compared to the same cells grown in the presence of empty vector-transfected media.The apoptosis rate with NPTX2-conditioned media(41.64±1.07,n=3) was significant higher than that of empty vector-transfected media(19.01±1.15,n=3) and that of DMEM median(13.32±1.00,n=3).The recombinant GST-NPTX2 was successfully expressed.However,the recombinant GST-NPTX2 showed no effect on the cell apoptosis and growth.Consults:The NPTX2 gene had the pro-apoptotic effect on pancreatic cancer cells.It was hypothesized that secreted NPTX2 was responsible for the growth inhibition.Neither recombinant GST nor GST-NPTX2 protein had any effect on apoptosis of the cells,which suggested that either post-translational modifications or an accessible amino-terminus might be required for NPTX2 to function.In conclusion:1.The hypermethylation of the promoter of ELA3B gene was one of the reasons that resulted in the down-regulation of ELA3B gene in pancreatic tissues.2.NPTX2 gene was down-regulated in human pancreatic cancer tissues.3.The hypermethylation of NPTX2 gene promoter played an important role of the down-regulation in pancreatic cancer.4.Successfully construct the eukaryotic and prokaryotic expression vectors of NPTX2 gene.5.The NPTX2 gene had the pro-apoptotic effect on pancreatic cancer cells.Either post-translational modifications or an accessible amino-terminus might be required for NPTX2 to function.