PRDM5基因抑制前列腺癌细胞22Rv1生长

目的 探讨PRDM5基因在前列腺癌细胞中的抑癌作用。方法 采用聚合酶链反应(PCR)、限制性内切酶酶切、T4 DNA连接酶连接等方法,克隆PRDM5基因并插入慢病毒载体中。将携带PRDM5基因的慢病毒质粒(或阴性对照质粒)与慢病毒包装质粒通过脂质体法共转染293T细胞,收集病毒上清,感染人前列腺癌细胞株22Rv1。用蛋白质印迹法检测细胞 PRDM5的表达,细胞倍增实验和平板克隆实验检测细胞增殖和克隆形成能力,软琼脂克隆形成实验检测细胞非锚着依赖性生长能力。结果 成功构建PRDM5重组慢病毒载体,并包装获得慢病毒上清。将PRDM5重组慢病毒载体感染22Rv1细胞后,筛选得到稳定表达细胞株,蛋白质印迹法结果显示该细胞株能稳定表达外源性PRDM5蛋白。过表达PRDM5的前列腺癌22Rv1细胞的增殖能力[倍增时间:(52.5±1.4) h vs (44.0±1.3) h]、克隆形成能力[克隆形成数: (1 114±98)/皿 vs (1 361±123)/皿]和非锚着依赖性生长能力[克隆形成数:(94.6±8.7)/孔 vs (154.0±3.5)/孔]均低于阴性对照组(P<0.05)。结论 前列腺癌22Rv1细胞中PRDM5的过表达具有抑制肿瘤细胞增殖、克隆形成和非锚着依赖性生长的能力。
Objective To investigate the antitumor effect of PRDM5 gene in prostate cancer cells. Methods PRDM5 gene was cloned and inserted into lentiviral vector using polymerase chain reaction (PCR), restriction endonuclease and T4 DNA ligase connected method. The lentiviral plasmids carrying PRDM5 gene (LV-PRDM5) or control lentivirus (LV-Luc) were co-transfected with lentiviral packaging plasmid mix into 293T cells by liposome method. The viral supernatants were collected and transduced into human prostate cancer cells 22Rv1. The expression of PRDM5 was verified by Western blotting analysis. The cell proliferation and clone formation ability were detected by cell multiplication and cell cloning experiments. The anchorage independent growth rate of prostate cancer cells was assessed by soft agar colony formation assay. Results The lentivirus vector expressing PRDM5 gene was constructed successfully, and the viral supernatants were obtained. The prostate cancer cell line 22Rv1 stably expressing exogenous PRDM5 was screened and verified by Western blotting analysis. Compared with control cells, the prostate cancer cell line 22Rv1 expressing PRDM5 showed a lower growth rate (multiplication time: [52.5±1.4] vs [44.0±1.3] h), clone formation rate ([1 114±98] vs [1 361±123] colonies per dish) and anchorage independent growth rate ([94.6±8.7] vs [154.0±3.5] colonies per cell, P<0.05). Conclusion Overexpression of PRDM5 has inhibitory effect against proliferation, clone formation and anchorage independent growth of prostate cancer cells in vitro