| Castration-resistant prostate cancer is more difficult for the treatment than hormone-sensitive prostate cancer,and the prognosis is poor.Therefore,it is necessary to develop new treatment methods and gene therapy is currently the most popular method for the treatment of malignant tumors,causing widespread concern among researchers.Gene therapy is to introduce the target gene into the target cell with a suitable vector,repair the related gene defects,and achieve the purpose of treating the tumors.This study aimed to construct a conditionally replicating adenovirus(hereinafter referred to as adenovirus)regulated by human telomerase catalytic subunit and enhancer of zeste homolog 2-short hairpin RNA(hereinafter referred to as EZH2-shRNA),which targeting inhibits the proliferation and invasion of CRPC cells.The goal of safe,efficient and stable treatment of CRPC provides a experimental basis for gene-target therapy of CRPC.Objective:To detect the expression of EZH2 protein in human prostate tissues and construct adenovirus regulated by hTERT and EZH2-shRNA,exploring the effect of adenovirus on the proliferation and invasion of CRPC cells,which provided a experimental basis for gene targeting therapy of CRPC.Methods:Prostate tissue samples of CRPC,BPH and HSPC were obtained.Tissue samples were selected to detect the expression of EZH2 by immunohistochemistry.A conditionally replicated adenovirus regulated by hTERT and EZH2-shRNA was constructed.Two shRNAs(shRNA1 and shRNA2)were designed.Cell counting kit-8 assay was used to detect the interference efficiency of adenovirus containing two different shRNAs on the proliferation of PC-3 cells.ShRNA with better interference effect was selected for subsequent experiments.Adenovirus with better interference effect was used to transfect human lung adenocarcinoma A549 cells.The fluorescence image of the transfected cells was observed under the microscope to observe whether the adenovirus was successfully constructed.PCa cells(PC-3 and DU145)were transfected with screened adenovirus.CCK-8 and transwell were used to detect the proliferation and invasion ability of PC-3 and DU 145 cells before and after transfection.Western blot was used to detect the protein expression of EZH2,CCND1,Ki-67 and E-cadherin in PC-3 and DU 145 cells before and after adenovirus transfection.Results:1.Immunohistochemistry showed:Compared with BPH specimens,the expression of EZH2 was significantly up-regulated in HSPC and CRPC patients.The difference was statistically significant(P<0.05).At the same time,in different types of PCa specimens(CRPC and HSPC),the expression of EZH2 was scored.The proportion of HSPC in the lower grade was larger,while the proportion of CRPC in the higher grade was larger.The difference between the two groups was statistically significant(P<0.05).2.Gene sequencing proved the correctness of shRNA and hTERT sequences in monoclonal antibodies,indicating the successful construction of monoclonal vectors.CCK-8 assay was used to detect the interference efficiency of shRNA-1 and shRNA-2 on the proliferation of PC-3 cells.The results showed that shRNA-2 more effectively inhibited the proliferation of PC-3 cells.At the same time,we used adenovirus carrying EZH2-shRNA2 and hTERT gene to interfere with A549 cells.Fluorescence analysis after 24,48 and 72 h showed that the number of transfected cells increased gradually with time,which indirectly indicated that the adenovirus vector was successfully constructed.3.Transwell invasion experiment showed that after incubating,the experimental group(cell group transfected with adenovirus)could significantly inhibit the invasion ability of PC-3 and DU145 cells,indicating that Ad-hTERT-EZH2-shRNA could significantly inhibit the invasion ability of prostate cancer cells.There was significant difference between the experimental group and the negative control group(cell without adenovirus transfection)(P<0.05).4.Compared with the untransfected cells,the results of CCK-8 showed that adenovirus had a significant inhibitory effect on the proliferation of prostate cancer cells of PC-3 and DU145 cells after 48 h(P<0.05).5.Western blot showed that the protein expression of Ki-67 and CCND1 decreased when we inhibite the protein expression of EZH2.On the contrary,E-cadherin increased.Conclusion:1.The expression level of EZH2 protein in human PCa tissues was increased,and the expression of EZH2 protein in CRPC tissues was significantly up-regulated than HSPC.2.Compared with EZH2 intensity score,EZH2 protein was significantly different in HSPC and CRPC tissues.3.CRAds were successfully constructed and could stably expressed in PCa cells.4.Transfection of CRAds regulated by hTERT and EZH2-shRNA into human PCa cells can inhibit the proliferation and invasion of PCa cells,showing significant anti-tumor effects.5.The down-regulation of EZH2 protein expression resulted in the down-regulation of Ki-67 and CCND1 protein expression and the up-regulation of E-cadherin protein expression. |
