Effects And Related Mechanisms Of EZH2 On The Cell Malignant Proliferation In Pancreatic Cancer

The aim of this study was to explore the role of epigenetic factor EZH2 in the malignant proliferation of pancreatic cancer and to expound its molecular mechanism,which might provide novel therapic ideas and theoretical basis for pancreatic cancer.The expression of EZH2 is abnormally high in the development and progressive cancer,which disorders the proliferation,cycle,migration and invasion of cancer cells.In this study,we constructed the stable normal pancreatic cells and pancreatic cancer cells with the overexpression,knockdown and set domain deletion of EZH2,respectively.Then the biological characteristics of these cells were detected and discussed.The related molecular mechanism was explored through transcriptome sequencing,which could provide important theoretical and experimental basis for new diagnostic markers and new drug targets in pancreatic cancer.The main contents were as follows.1.60 paraffin sections of pancreatic cancer and their clinical data were collected.The expression of EZH2 in different patients was clarified by immunohistochemistry.The results showed that EZH2 was highly expressed in nuclei of pancreatic cancer cells,which were pale yellow to brownish granules.The normal pancreatic tissue was blue,suggesting that the expression of EZH2 was not observed.2.The overexpression and set domain deletion plasmids of EZH2 gene were constructed.The knockdown plasmids of EZH2 gene were purchased.Then they were verified by PCR and sequencing.The positive plasmids were used to transfected into normal pancreatic cell HPDE6-C7 and pancreatic cancer cell BxPC-3 with lentiviral transfection method.Then the expression of EZH2 were detected by RT-qPCR and Western blot.The results showed that we successfully constructed these stable cells.3.The proliferation of normal pancreatic cells and pancreatic cancer cells was determined by MTS assay,colony forming assay and Ki-67 antibody test.The results showed that transfected EZH2 overexpression plasmid could enhance the proliferation ability of BxPC-3 cells,and enhanced 38% compared with the control.Transfection of EZH2 knockdown plasmids decreased cell proliferation and the decrease range was 16% to 40% compared with the control.After deletion of the setdomain region of EZH2,the proliferation capacity of the cells was not change.4.The cell cycle of normal pancreatic cells and pancreatic cancer cells was determined by flow cytometry.The results showed that EZH2 had little influence on cell cycle of pancreatic cancer cell BxPC-3.5.The migration of normal pancreatic cells and pancreatic cancer cells was examined by scratch test and Transwell test.The results showed that EZH2 overexpression could significantly enhance the migration ability of cells.EZH2 knockdown weakened the migration ability of cells.When EZH2 set domain deleted,the cell migration ability had not been influenced.6.The EZH2 gene knockdown plasmid and its empty vector were used for transcriptome sequencing.According to the results of bioinformatics analysis,four DEGs(E2F1,GLI1,CDK3 and Mcm4)associated with cell proliferation were selected for RT-qPCR.The results were consistent with the expression levels of transcriptional sequencing,indicating that EZH2 may affect cell proliferation and relate with them.In addition,the marker of epithelial-mesenchymal transition(EMT)E-cadherin(CDH1)expression was lower in EZH2 overexpression cells than control,while E-cadherin expression was higher in EZH2 knockdown cells than control.This is consistent with the effect of EZH2 on cell migration.These results suggested that EZH2 might play an important role in the pathogenesis and invasion of pancreatic carcinoma through regulating E-cadherin.These preliminarily expounded the molecule mechanism of EZH2 on the cell malignant proliferation of pancreatic cancer.