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目的:构建GPRC6A基因过表达的前列腺癌LncapC4-2细胞株,检测细胞迁移和侵袭能力改变及EMT相关基因表达,为研究GPRC6A介导的EMT在前列腺癌发生发展过程中的作用奠定基础。方法:构建过表达GPRC6A的慢病毒载体pCDH-GPRC6A,人胚肾293T细胞包被病毒并感染Lncap C4-2细胞株,Puromycin筛选获得稳定过表达GPRC6A的细胞株。RT-PCR和Western blot验证细胞GPRC6A的过表达情况。划痕和transwell实验检测细胞迁移和侵袭能力,qPCR检测EMT相关基因表达。结果:成功构建GPRC6A表达的慢病毒载体,RT-PCR和Western blot检测LncapC4-2 GPRC6A过表达细胞株中GPRC6A的mRNA和蛋白质表达增高,与对照相比较,在过表达GPRC6A的LncapC4-2细胞中,细胞迁移和侵袭能力增强,并且EMT相关基因E-cadherin表达降低而SNAIL表达增高。结论:成功构建过表达的GPRC6A的前列腺癌细胞株,肿瘤细胞中的GPRC6A可能通过增强细胞迁移和侵袭能力并促进细胞EMT而参与前列腺癌发生发展过程。
OBJECTIVE: To construct GPR6A-overexpressing prostate cancer LncapC4-2 cell line to detect the changes of cell migration and invasion and the expression of EMT-related genes, so as to lay the foundation for the study of GPRC6A-mediated EMT in the development and progression of prostate cancer. Methods: The lentiviral vector pCDH-GPRC6A overexpressing GPRC6A was constructed. Human embryonic kidney 293T cells were infected with the virus and infected with Lncap C4-2 cells. Puromycin cells were selected and stably overexpressed GPRC6A. The overexpression of GPRC6A was verified by RT-PCR and Western blot. Scratches and transwell assays were used to detect cell migration and invasion, and qPCR was used to detect EMT-related gene expression. Results: The GPRC6A expression lentiviral vector was successfully constructed. The mRNA and protein expression of GPRC6A in LNCaCLC4-2 GPRC6A overexpressing cell line was detected by RT-PCR and Western blot. Compared with the control, GPRC6A overexpression in LncapC4-2 cells , Enhanced cell migration and invasion, and decreased the expression of EMT-related genes E-cadherin and SNAIL. CONCLUSION: GPRC6A overexpressed in overexpressed GPRC6A cells may be involved in the development of prostate cancer by enhancing cell migration and invasion and promoting EMT.