摘要目的 探讨p21活化激酶4(PAK4)对乳腺癌细胞增殖和干细胞特性的影响及其作用机制.方法:采用乳腺癌MCF-7细胞构建短发卡RNA (shRNA)-NC和shRNA-PAK4慢病毒稳定细胞系,采用实时定量聚合酶链反应(Real-time PCR)检测PAK4 mRNA表达水平;采用细胞计数试剂盒(CCK-8)法检测细胞增殖;采用Transwell分析细胞侵袭能力;采用微球体形成实验分析乳腺癌细胞的干细胞特性;采用蛋白质印迹法(Western blot)检测乳腺癌干细胞标志分子乙醛脱氢酶1(ALDH1)和Oct-4的表达.结果 与shRNA-NC细胞比较(1.71±0.14),shRNA-PAK4细胞中PAK4的mRNA表达水平明显下调(0.57±0.05),差异有统计学意义(t=3.725,P＜0.05);与shRNA-NC细胞比较(1.76±0.14),shRNA-PAK4细胞的增殖能力显著下降(0.66±0.04),差异有统计学意义(t=3.572,P＜0.05);Transwell结果显示shRNA-PAK4细胞的侵袭能力(58.06±7.73)较shRNA-NC细胞(92.18±11.35)显著下降,差异有统计学意义(t=2.850,P＜0.05);Western blot结果显示,与shRNA-NC细胞比较(1.48±0.12、2.16 ±0.18),shRNA-PAK4细胞中ALDH1和Oct-4的蛋白表达水平明显下降(0.40 ±0.04、0.77 ±0.06),差异有统计学意义(t=4.361、3.505,P＜0.05).结论 PAK4可通过正向调控ALDH1和Oct-4的表达改变乳腺癌细胞的干细胞特性,从而参与乳腺癌细胞的增殖和侵袭.

abstractsObjective To explore the effects of p21-activated protein kinase 4 (PAK4) on proliferation and stem cell characteristics of breast cancer cells and its possible mechanism.Methods The short hairpin RNA (shRNA)-NC and shRNA-PAK4 lentiviral stable cell lines were constructed using MCF-7 cells,and the PAK4 mRNA expression was detected by real-time quantitative polymerase chain reaction (Real-time PCR).Cell proliferation was detected by cell counting kit-8 (CCK-8),and cell invasion ability was analyzed by Transwell.The stem cell characteristics of breast cancer cells was analyzed by microsphere formation assay.Western blotting was used to detect the expression of aldehyde dehydrogenase 1 (ALDH1) and Oct-4.Results Compared with shRNA-NC cells (1.71 ± 0.14),the mRNA expression of PAK4 in shRNA-PAK4 cells (0.57 ± 0.05) was significantly down-regulated (t =3.725,P ＜ 0.05).Compared with shRNA-NC cells (1.76 ± 0.14),the proliferation ability of shRNA-PAK4 cells (0.66 ± 0.04) decreased significantly (t =3.572,P ＜ 0.05).Transwell showed that the invasive ability of shRNA-PAK4 cells (58.06 ± 7.73) was significantly lower than that of shRNA-NC cells (92.18 ± 11.35) (t =2.850,P ＜ 0.05).Western blotting showed that compared with shRNA-NC cells (1.48 ±0.12,2.16 ±0.18),the expression of ALDH I and Oct-4 in shRNA-PAK4 cells (0.40 ±0.04,0.77±0.06) were both decreased significantly (t=4.361,3.505,P＜0.05).Conclusion PAK4 regulates the stem cell characteristics of breast cancer cells by positively regulating the expression of ALDH I and Oct-4,thus participating in the proliferation and invasion of breast cancer cells.