p21活化激酶4蛋白对乳腺癌细胞凋亡、迁移侵袭和上皮-间充质转化的影响

Effect of p-21 activated protein kinase protein on apoptosis,migration and invasion and epithelial-mesenchymal transition of breast cancer cells

目的:探讨p21活化激酶4(PAK4)蛋白对乳腺癌细胞凋亡、侵袭迁移和上皮-间充质转化(EMT)的影响。方法:选取2018年7月至2019年7月河南省人民医院收治的175例乳腺癌和癌旁组织作为研究对象,采用免疫组织化学分析PAK4蛋白表达水平。MCF-7细胞随机分为两组,采用慢病毒介导PAK4短发卡RNA(shRNA)和对照shRNA感染细胞,分别为PAK4 KD组和对照组。采用膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)凋亡检测试剂盒和TUNEL染色分析两组细胞凋亡水平;采用划痕实验分析两组细胞迁移;采用Transwell分析两组细胞侵袭能力;采用蛋白质印迹法(Western blot)分析两组上皮细胞标志物E-钙黏蛋白(E-cadherin)、β-连环素(β-catenin)和间充质标志物波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)表达水平。应用SPSS 17.0统计学软件分析,组间比较采用t检验。结果:癌旁组织PAK4平均阳性面积(72.49±8.90)明显低于乳腺癌肿瘤组织PAK4平均阳性面积(258.12±29.21),差异有统计学意义(t=8.190,P<0.05)。对照组乳腺癌细胞凋亡比例[(2.81±0.94)%]明显低于PAK4 KD组乳腺癌细胞凋亡比例[(19.36±4.27)%],差异有统计学意义(t=5.109,P<0.05)。对照组乳腺癌细胞TUNEL染色阳性率[(4.31±0.86)%]明显低于PAK4 KD组乳腺癌细胞TUNEL染色阳性率[(24.21±6.02)%],差异有统计学意义(t=7.119,P<0.05)。对照组细胞划痕愈合率[(90.45±8.92)%]明显高于PAK4 KD组乳腺癌细胞划痕愈合率[(53.44±6.18)%],差异有统计学意义(t=10.312,P<0.05)。对照组细胞侵袭数量[(154.22±14.38)个]明显高于PAK4 KD组乳腺癌细胞侵袭数量[(89.23±9.02)个],差异有统计学意义(t=9.112,P<0.05)。对照组细胞上皮细胞表型标志物E-cadherin表达水平(0.61±0.21)明显低于PAK4 KD组乳腺癌细胞(1.24±0.28),差异有统计学意义(t=3.981,P<0.05)。对照组细胞间质细胞表型标志物Vimentin和N-cadherin表达水平(1.77±0.31、1.29±0.19)明显高于PAK4 KD组乳腺癌细胞(1.02±0.21、0.79±0.15),差异有统计学意义(t=3.209、3.001,P<0.05)。结论:PAK4蛋白在乳腺癌组织中呈高表达,参与乳腺癌细胞凋亡、迁移、侵袭和EMT等过程。

Objective To investigate the effect of p-21 activated protein kinase(PAK4)on apoptosis,invasion and migration and epithelial-mesenchymal transition(EMT)of breast cancer cells.Methods Totally,175 cases of breast cancer and adjacent tissues were selected as the study subjects from July 2018 to July 2019.The expression level of PAK4 protein was analyzed by immunohistochemistry.MCF-7 cells were randomly divided into two groups.The infection of PAK4 shRNA and control short hairpin RNA(shRNA)was mediated by lentivirus,respectively,named PAK4 KD group and control group.The apoptosis levels of the two groups were analyzed by annexin V-fluoresceine isothiocyanate(Annexin V-FITC)and TUNEL staining.The migration of the two groups was analyzed by scratch test;The invasion ability of the two groups was analyzed by Transwell.The E-cadherin,vimentin and N-cadherin were analyzed by Western blotting.SPSS 17.0 statistical software was used to analyze the data and t test was used for comparison between groups.Results The average positive area of PAK4 in the adjacent tissues(72.49±8.90)was significantly lower than that of breast cancer tissues(258.12±29.21,t=8.190,P<0.05).The apoptosis rate in control group[(2.81±0.94)%]was significantly lower than that in PAK4 KD group[(19.36±4.27)%,t=5.109,P<0.05].The positive rate of TUNEL staining in control group[(4.31±0.86)%]was significantly lower than that in PAK4 KD group[(24.21±6.02)%,t=7.119,P<0.05].The rate of scratch healing in control group[(90.45±8.92)%]was significantly higher than that in PAK4 KD group[(53.44±6.18)%,t=10.312,P<0.05].The number of invasive cells in control group[(154.22±14.38)]was significantly greater than that in PAK4 KD group[(89.23±9.02),t=9.112,P<0.05].The expression level of E-cadherin in control group(0.61±0.21)was significantly lower than that in PAK4 KD group[(1.24±0.28),t=3.981,P<0.05].The expression levels of vimentin and N-cadherin in control group[(1.77±0.31),(1.29±0.19)]were significantly higher than those in PAK4 KD group[(1.02±0.21),(0.79±0.15),t=3.209,3.001,P<0.05].Conclusion PAK4 protein is highly expressed in breast cancer tissues,and it is involved in the apoptosis,migration,invasion and EMT of breast cancer cells.