Annexin A5沉默对ERK1/2 MAPK-Bax凋亡通路影响和意义

Annexin A5 silence for the influence and significance of ERK1/2MAPK-Bax apoptosis pathway

目的探讨结肠癌细胞株RKO中Annexin A5对ERK1/2 MAPK-Bax凋亡通路的影响。方法慢病毒转染人结肠癌RKO细胞后进行稳定细胞株筛选;用流式凋亡和Hoechst 33258实验探究Annexin A5沉默前后的细胞株凋亡情况;采用RT-PCR法及蛋白质印迹法验证Annexin A5沉默前后ERK1/2、Bax和Bcl-2的mRNA与蛋白表达量的变化。结果实验组相对于空白对照组Annexin A5的mRNA相对表达量从1.010±0.017下降到0.056±0.005,下降了(95.34±1.54)%,F=2 320.600,P<0.000 1。Annexin A5沉默前后的细胞凋亡率分别为(24.87±6.18)%和(10.7±2.66)%,沉默后凋亡率较之前明显下降,t=3.57,P=0.023。Annexin A5沉默后Bax的mRNA相对表达量从1.000±0.000下降到0.732±0.034,下降了(26.79±3.41)%,F=9.884,P<0.05;但蛋白相对表达量无明显变化,P=0.945;Bcl-2mRNA相对表达量从1.000±0.000上升到1.592±0.085,上升了(59.17±8.49)%,F=84.026,P<0.001;蛋白相对表达量从0.671±0.018上升到1.580±0.019上升了(135.78±8.89)%,F=2 195.872,P<0.000 1。磷酸化后的ERK1/2蛋白相对表达量从0.917±0.062上升到1.041±0.045,上升了(13.75±5.88)%,F=6.948,P<0.05。结论Annexin A5本身可能影响或参与了肿瘤细胞的凋亡过程,ERK1/2 MAPK凋亡通路可能是其影响或参与机制之一。

OBJECTIVE To investigate the influence of Annexin A5 in colon cancer cell line RKO on ERK1/2MAPK-Bax apoptotic pathway.METHODS Stable cell line of RKO colon cancer cells was selected after lentiviral transfection.Flow cytometry apoptosis and Hoechst 33258 experiments were used to explore the Annexin A5 silence before and after the apoptosis of cell lines.By RT-PCR and western blot method to validate ERK1/2,Bax,Bcl-2 mRNA and protein expression quantity of the change of the Annexin A5 silence before and after.RESULTS In experimental group compared with the Blank group Annexin A5 mRNA relative expression decreased（95.34±1.54）% from 1.010±0.017 to 0.056±0.005（F=2 320.600,P〈0.000 1）.Annexin A5 silence before and after the apoptosis rate was（24.87±6.18）%,（10.70±2.66）%,the apoptotic rate decreased significantly after silence（t=3.57,P=0.023）.Bax mRNA relative expression decreased（26.79±3.41）%from 1.000±0.000 to 0.732±0.034 after Annexin A5silence（F=9.884,P〈0.05）.But had no obvious changes of protein relative expression（P=0.945）.The Bcl-2mRNA relative expression rose（59.17%±8.49）%from 1.000±0.000 to 1.592±0.085（F=84.026,P〈0.001）.Protein relative expression rose（135.78±8.89）%from 0.671±0.018 to 1.580±0.019（F=2 195.872,P〈0.000 1）.After the phosphorylation of ERK1/2protein relative expression increased（13.75±5.88）% from 0.917±0.062 to 1.041±0.045（F=6.948,P〈0.05）.CONCLUSION Annexin A5 may affect or participate in the process of the tumor cell apoptosis,ERK1/2 MAPK apoptosis pathway may be the impact or participate in one of the mechanisms.