目的:研究肝细胞黏附分子(hepatocyte cell adhesion molecule,hepaCAM)与藏花素联用对前列腺癌(prostate cancer,PCa)细胞DU145增殖的影响。方法:四甲基偶氮唑盐(methylthiazolyldiphenyl-tetrazolium bromide,MTT)法检测细胞增殖效应;克隆形成实验检测细胞克隆形成率;实时荧光定量PCR、Western blot法检测细胞周期调节因子cyclinD1及增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达变化。结果:hepaCAM过表达腺病毒与藏花素单独使用均能抑制PCa细胞DU145的增殖[48 h抑制率分别为(12.0±2.0)%,(22.0±1.0)%],但两者联用比单独应用效果更加明显[48 h抑制率为(54.0±1.0)%,P=0.000]。Real-time PCR结果显示,hepaCAM过表达腺病毒及藏花素联合作用与藏花素单独使用相比,cyclinD1 mRNA表达下调(P=0.048);两者联合应用与hepaCAM过表达腺病毒或藏花素单独使用相比,PCNA mRNA水平下调(P=0.047,P=0.006)。Western blot结果显示,与单独使用hepaCAM过表达腺病毒或藏花素相比,两者联用组cyclinD1蛋白表达明显降低(P=0.002,P=0.002),同时PCNA蛋白表达也降低(P=0.000,P=0.003)。结论:hepaCAM基因过表达腺病毒与藏花素联用对PCa DU145细胞增殖的抑制有明显的协同作用,作用机制与下调cyclinD1和PCNA的表达有关。
Objective:To investigate the effects of hepatocyte cell adhesion molecule(hepaCAM)overexpression adenovirus combined with crocin on cell proliferation in prostate cancer cell line DU145. Methods:MTT assay was used to evaluate the effect of cell proliferation. Colony formation test was used to measure colony formation rate of single cell. Real-time quantitative PCR(qPCR)assay and Western blot were used to detect mRNA and protein expression of cyclinD1 and PCNA respectively. Results:After being treated with hepaCAM overexpression adenovirus or crocin alone,the proliferation of DU145 cell was inhibited[IR=(12.0±2.0)%,(22.0±1.0)%],but the effect was more remarkable when the cells being treated with hepaCAM overexpression adenovirus and crocin com-bined[IR=(54.0±1.0)%,P=0.000]. Expression of cyclinD1 mRNA was down-regulated after being treated with hepaCAM overexpression adenovirus and crocin combined compared with being treated with crocin alone detected by qPCR(P=0.048),and proliferating cell nuclear antigen(PCNA)mRNA was down-regulated after being treated with hepaCAM overexpression adenovirus and crocin combined compared with being treated with hepaCAM overexpression adenovirus or crocin alone(P=0.047,P=0.006). Western blot showed that compared with hepaCAM group or crocin group,expressin of cyclinD1 protein was down-regulated in hepaCAM and crocin combined group(P=0.002,P=0.002),meanwhile expression of PCNA protein was also down-regulated(P=0.000,P=0.003).Conclusion:hepaCAM overexpression adenovirus combined with crocin can inhibit cell proliferation of prostate cancer cell line DU145 and the machanism is associated with down-regulation of cyclinD1 and PCNA expression.