GPRC6A过表达前列腺癌细胞株构建及EMT相关研究

doi:10.13523/j.cb.20170303

中国生物工程杂志

2017, Vol. 37

Issue (3): 18-26 DOI: 10.13523/j.cb.20170303

研究报告

GPRC6A过表达前列腺癌细胞株构建及EMT相关研究

盛彬¹, 杨帆², 孙心旖¹, 陈瑶¹, 李莉¹, 杨建一¹, 杜圣家¹, 刘铭¹

1. 山西医科大学基础医学院太原 030001;
2. 北京协和医学院研究生院北京 100005

Construction of the Stable Prostate Cancer Cell Lines Overexpress GPRC6A and EMT Study

SHENG Bin¹, YANG Fan², SUN Xin-yi¹, CHEN Yao¹, LI Li¹, YANG Jian-yi¹, DU Sheng-jia¹, LIU Ming¹

1. School of Basic Medical Science, Shanxi Medical University, Taiyuan 030001, China;
2. Peking Union Medical College and Chinese Academy of Medical Sciences, Graduate School, Beijing 100005, China

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摘要：

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LncapC4-2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。

关键词： 前列腺癌; 上皮间质转化; GPRC6A

Abstract:

Objective:To construct the prostate cancer cell line LncapC4-2 that overexpress GPRC6A gene, detect the cell migration and invasion abilities and the expression of EMT related genes, thus to establish the basis of exploring the role of EMT induced by GPRC6A in prostate cancer progression. Methods:The GPRC6A overexpression lentivirus vector pCDH-GPRC6A was constructed, The lentivirus were packaged using human embryonic kidney 293T cells and LncapC4-2 cell line was infected, and GPRC6A stably overexpressing cell line was screened using puromycin. RT-qPCR and Western blot methods were used to verify the overexpression of GPRC6A in the cell line, scratches and transwell cell migration assay were used to test the cell migration and invasion abilities and QPCR was used to detect the expressions of EMT-associated genes. Results:The lentivirus vector pCDH-GPRC6A was constructed successfully. In GPRC6A overexpression Lncap C4-2 cells, the GPRC6A expression was increased in mRNA and protein levels detected by using RT-PCR and Western blot methods. The cell migration and invasion abilities were higher in GPRC6A overexpression Lncap C4-2 cells than those in control cells. The expressions of EMT-associated gene E-cadherin and SNAIL were reduced and increased, respectively, in GPRC6A overexpression LncapC4-2 cells. Conclusion:The prostate cancer cell line LncapC4-2 that stably overexpress GPRC6A has been constructed successfully. GPRC6A maybe involves in the progression of prostate cancer by enhancing the abilities of cell migration and invasion, and promoting EMT in cancer cells.

Key words: GPRC6A EMT Prostate cancer

收稿日期: 2016-09-29 出版日期: 2017-03-25

ZTFLH:

Q813

基金资助:

国家青年科学基金（81302220），山西省青年科学基金（2014021037-1），山西医科大学博士启动基金（B03201204）资助项目

通讯作者: 刘铭 E-mail: lium0421@163.com

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引用本文:

盛彬, 杨帆, 孙心旖, 陈瑶, 李莉, 杨建一, 杜圣家, 刘铭. GPRC6A过表达前列腺癌细胞株构建及EMT相关研究[J]. 中国生物工程杂志, 2017, 37(3): 18-26.

SHENG Bin, YANG Fan, SUN Xin-yi, CHEN Yao, LI Li, YANG Jian-yi, DU Sheng-jia, LIU Ming. Construction of the Stable Prostate Cancer Cell Lines Overexpress GPRC6A and EMT Study. China Biotechnology, 2017, 37(3): 18-26.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170303 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I3/18

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