蛋白质精氨酸甲基转移酶5促进人卵巢癌HO8910细胞迁移

Protein arginine methyltransferase 5 promotes the migration of human ovarian cancer HO8910 cells

目的:探讨蛋白质精氨酸甲基转移酶5(protein arginine methyltransferase 5,PRMT5)对人卵巢癌HO8910细胞上皮间质转化(epithelial-to-mesenchymal transition,EMT)和迁移能力的影响。方法:利用瞬时转染方法建立PRMT5过表达的HO8910细胞株,设空白对照组(野生型HO8910细胞株)、阴性对照组(转染pCMV-myc质粒)、实验组(转染pCMV-Myc-PRMT5质粒);同时采用shRNA干扰方法建立诱导型稳定敲低PRMT5基因的HO8910细胞株,设pLKO对照组(感染空载体慢病毒)、pKLO+Dox(100 ng/mL)组、shPRMT5组(感染PRMT5 shRNA慢病毒)和shPRMT5+Dox(100 ng/mL)组;通过免疫印迹和qRT-PCR实验检测PRMT5 mRNA表达和干扰效率。实验分为shPRMT5组、shPRMT5+Dox组、pCMV-Myc组、pCMV-Myc-PRMT5组;Transwell实验检测细胞迁移能力;免疫印迹法检测EMT相关蛋白表达。结果:免疫印迹和qRT-PCR验证成功建立过表达PRMT5和稳定诱导型敲低PRMT5的卵巢癌HO8910细胞株。PRMT5敲低后,卵巢癌HO8910细胞迁移能力显著下降(P均<0.01),间质标志相关分子包括基质金属蛋白酶2,波形蛋白,N-钙黏蛋白表达下调,上皮标志分子E-钙黏蛋白表达上调。此外,在卵巢癌细胞中外源表达PRMT5可显著促进HO8910细胞迁移(P均<0.05)。结论:PRMT5能够促进卵巢癌HO8910细胞EMT进程,促进细胞迁移。

Objective:To investigate the effect of protein arginine methyltransferase 5(PRMT5)on epithelial-to-mesenchymal transition(EMT)and migration ability in human ovarian cancer HO8910 cells.Methods:PRMT5 overexpressed HO8910 cell line was established by transient transfection method,and divided into blank control group(wild-type HO8910 cell line),negative control group(HO8910 cells transfected with pCMV-Myc plasmid),and experimental group(HO8910 cells transfected with pCMV-Myc-PRMT5 plasmid).At the same time,HO8910 cell lines with stable PRMT5 knockdown were established by shRNA interference and divided into pLKO control group(infected with empty lentivirus),pKLO+Dox group(100 ng/mL),shPRMT5 group(infected with PRMT5 shRNA lentivirus)and shPRMT5+Dox group(100 ng/mL).The mRNA expression and interference efficiency of PRMT5 were detected by Western blotting or qRT-PCR.The experiment was divided into shPRMT5 group,shPRMT5+Dox group,pCMV-Myc group and pCMV-Myc-PRMT5 group;Transwell assay was used to detect the cell migration ability.The expression of EMT-related proteins was detected by Western blotting.Results:Western blotting and qRT-PCR verify the establishment of ovarian cancer HO8910 cell lines with PRMT5 overexpression and stable knockdown PRMT5.After PRMT5 knockdown,the mobility of ovarian cancer HO8910 cells was significantly decreased(all P<0.01),and the expression of mesenchymal marker related molecules including matrix metalloproteinase-2,vimentin,and N-cadherin was down-regulated,and the expression of epithelial marker E-cadherin was up-regulated.In addition,exogenous PRMT5 expression in ovarian cancer cells greatly up-regulated the migration ability of HO8910 cells(all P<0.05).Conclusion:PRMT5 could promote the EMT process of ovarian cancer HO8910 cells and enhance cell migration.