Study On The Effect And Mechanisms Of ME3 On The Proliferation,invasion And Metastasis In Pancreatic Cancer

PartⅠ Clinical significance of ME3 expression in pancreatic cancer tissuesObjective:To analyze the expression of ME3 in pancreatic cancer and its relationship with the prognosis of pancreatic cancer patients through data mining of various bioinformatics databases and detect the expression of ME3 gene in pancreatic cancer tissues.To provide a preliminary basis for the involvement of ME3 gene in the development of pancreatic cancer and the potential target of pancreatic cancer.Methods:The expression of ME3 in pancreatic cancer was analyzed by the Oncomine database.GEPIA database was used to analyze the difference in expression of ME3 gene in pancreatic cancer and normal pancreatic tissue.The relationship between the expression of ME3 mRNA and the prognosis in pancreatic cancer patients was analyzed in GDC database.The expression of ME3 in pancreatic cancer tissues and paracancerous tissues was detected by immunohistochemistry,quantitative PCR,and western blot.The differences of ME3 expression in pancreatic cancer tissues and paracancerous tissues were analyzed,and the correlation with the clinicopathological features and P53,Ki67 LI in pancreatic cancer patients were also analyzed.Meanwhile,the expression of ME3 mRNA and protein in pancreatic cancer tissues was dectected by the Real-time PCR and Western blot.Results:78 samples were extracted from Badea Pancreas dataset of the Oncomine database.It was found that the mRNA expression of ME3 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues(P<0.01).The expression of ME3 gene mRNA in 179 cases of pancreatic cancer and 171 normal pancreas tissues was extracted from TCGA dataset of GEPIA database.The results showed that the expression of ME3 gene in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues(P<0.05).In the GDC database,the PAAD data including ME3 high expression group(37 cases)and low expression group(47 cases)was extracted in the TCGA dataset.The overall survival rate of ME3 high expression group was significantly lower than that of ME3 low expression group with survival analysis(P<0.05).Immunohistochemistry was used to detect the expression of ME3 in pancreatic cancer and para-cancerous tissues.It was found that pancreatic cancer had a higher expression of ME3 than para-cancerous tissues(P<0.01).The expression of ME3 in pancreatic cancer was not related to gender and age(P>0.05),but was significantly correlated with differentiation,clinical stage and lymph node metastasis(P<0.05).The expression of ME3 was higher with the decrease of differentiation,progression of clinical stage and presence of lymph node metastasis,and the expression of ME3 was significantly correlated with the accumulation of P53 protein in pancreatic cancer(P<0.01).The expression of ME3 was positively correlated with Ki67 LI by the correlation analysis(P<0.01).At the same time,Real-time PCR and Western blot were used to detect the expression of ME3 in pancreatic cancer tissues and para-cancerous tissues.The expression of ME3 in pancreatic cancer tissues was significantly higher than in para-cancerous tissues(P<0.05)at both mRNA level and protein level.Conclusion:It was found that ME3 expression in pancreatic cancer was significantly higher than that in para-cancerous tissues,and expression of ME3 was closely related with clinical stage,differentiation,lymph node metastasis and activity of tumor proliferation,and was interacted with tumor suppressor gene(P53).It will provide a theoretical basis for the occurrence and development of pancreatic cancer,and further confirm that ME3 could be used as a new target in the pathogenesis of pancreatic cancer.It will be expected to provide new directions and ideas for gene therapy and targeted therapy of pancreatic cancer.Part Ⅱ The effect of ME3 on the biological function about proliferation,migration and invasion in pancreatic cancer cellObjective:To explore the role of ME3 gene in proliferation,migration and invasion of pancreatic cancer cells.Methods:The expression vectors of ME3 gene were constructed,and the effects of ME3 on proliferation,migration and invasion of pancreatic cancer cells were studied by CCK-8 assay,cell scratch test,Transwell migration and invasion assay.Results:ME3-shRNA plasmid was successfully constructed to down regulate/interfere with ME3 expression and Flag-ME3 plasmid was used to up regulate ME3 expression.The efficiency of the expression vector was detected and verified by real time PCR and Western blot.The effect of ME3 on the proliferation of pancreatic cancer was detected by CCK-8 assay.It was found that overexpression of ME3 in pancreatic cancer cells could promote the proliferation of pancreatic cancer cells,and down regulation of ME3 expression could inhibit the proliferation of pancreatic cancer cells.In addition,the expression of ME3 in pancreatic cancer cells was increased,the expression of CDC20 increased synchronously.The expression of ME3 was downregulated,and the CDC20 protein also decreased synchronously.Cell scratch test showed that the expression of ME3 was up-regulated,the healing rate of pancreatic cancer cells was significantly higher than that of the control group(P<0.01),and the expression of ME3 in pancreatic cancer cells was down regulated,and the healing rate of pancreatic cancer cells was significantly lower than that of the control group(P<0.01).The migration ability of pancreatic cancer cells was detected by Transwell migration assay.The results showed that the number of migrating cells was significantly higher than that of the control group with the up-regulated expression of ME3(P<0.01).And the number of migrating cells was significantly lower than that of the control group with the down-regulated expression of ME3(P<0.01).At the same time,Transwell migration assay was used to detect the migration ability of pancreatic cancer cells.The results showed that the number of migrating cells was significantly higher than that of the control group with the up-regulated expression of ME3(P<0.01).And the number of migrating cells was significantly lower than that of the control group with the down-regulated expression of ME3(P<0.01).In addition,the level of mRNA and protein expression of MMP2 and MMP9 genes was increased in pancreatic cancer cells with overexpression of ME3.On the contrary,when the expression of ME3 in pancreatic cancer cells was downregulated,the level of mRNA and protein expression of MMP2 and MMP9 genes decreased significantly.Conclusion:ME3 gene may be involved in the biological function of pancreatic cancer cell in proliferation,migration and invasion.It may be one of the important genes that affect the biological behavior of pancreatic cancer cells.It may also be a potential therapeutic target for pancreatic cancer and present a theoretical foundation for the diagnosis and therapy of pancreatic cancer.Part Ⅲ Effect of ME3 on pancreatic cancer cell EMT and its molecular mechanismObjective:To reveal the role and potential molecular mechanism of ME3 in epithelial mesenchymal transition of pancreatic cancer cells.Methods:By up-regulating and down-regulating the expression of ME3 gene,the expression of mRNA and protein levels of EMT related markers was detected by Realtime PCR and Western blot.And the expression of TGF-β/Smad signal pathway related genes was detected at the same time,and the relationship between ME3 expression and EMT,TGF-β/Smad signaling pathway were analyzed.The effect of EMT and migration and invasion of pancreatic cancer cells was analyzed after TGF-β stimulation.and after down-regulation of ME3 gene expression,the effect of EMT induced by TGF-β also was analyzed.Results:Transfection of Flag-ME3 plasmid and ME3-shRNA plasmid respectively upregulated and downregulated the expression of ME3 in pancreatic cancer cells.The expression of EMT related genes was detected by Real-time PCR and Western blot.It was found that the expression of N-cadherin,Vimentin and Snail increased significantly in the mRNA and protein level,but the expression of E-cadherin decreased.On the contrary,by down-regulating ME3 expression,the expression of E-cadherin increased evidently,while the expression of N-cadherin,Vimentin and Snail significantly decreased.Meanwhile,the expression of TGF-β/Smad signal pathway related genes was detected after up-regulation and down-regulation of ME3 expression.The results showed that when the expression of ME3 gene was up-regulated,the expression of Smad2 and Smad3 mRNA was only slightly higher than that of the control group,but the mRNA expression of TGF-βwas obviously higher than that in the control group.The expressions of Smad2/3,p-Smad2/3 and TGF-β protein were also higher than that in the control group.But there was a significant statistical difference between the two groups in the expression of p-Smad2/3 and TGF-β(P<0.05).When the expression of ME3 was down-regulated,the expression of Smad2 and Smad3 mRNA decreased only slightly,but the expression of TGF-β mRNA was significantly lower than that in the control group.And the expression of Smad2/3,p-Smad2/3 and TGF-β protein was also lower than those in the control group.There was significant difference between the two groups in the expression of p-Smad2/3 and TGF-β(P<0.05).At the mRNA level,whether ME3 expression was up-regulated or down-regulated,the expression of TGFβ was negatively correlated with E-cadherin.ME3 gene could promote the expression of TGF-β and inhibit the expression of E-cadherin,thereby promoting the initiation of EMT.After pancreatic cancer cells were treated with TGF-β,compared with the control group,the expression of E-cadherin decreased,and the expression of N-cadherin,Vimentin,and Snail increased.At the same time,the number of migrating and invading cells was significantly higher than that of the control group.Down-regulation of ME3 gene expression in pancreatic cancer cells,ME3-shRNA+TGF-β group compared with TGF-β group,E-cadherin expression increased,N-cadherin,Vimentin,Sail expression were all significantly decreased,P-Smad2/3 protein expression also dropped.Conclusion:ME3 can increase the EMT process of pancreatic cancer cells and promote the metastasis of pancreatic cancer through EMT.TGF-β/Smad signaling pathway may be a core pathway of EMT in pancreatic cancer.ME3 gene may promote the development of EMT in multiple ways through the regulation of TGF-β/Smad signaling pathway,and involve in the occurrence,development,invasion and metastasis of pancreatic cancer.So it will benefit to provide guidance for finding more effective intervention targets and new treatment strategies.