重组IL-12对人乳腺癌细胞自噬的研究

Study on Autophagy of Human Breast Cancer Cells by Recombinant IL-12

目的探讨重组人IL-12(rIL-12)对乳腺癌细胞自噬的影响。方法两株乳腺癌细胞(MDA-MB-231和MCF7)分别用rIL-12处理,经免疫蛋白印迹技术和细胞免疫荧光技术检测其自噬微管相关蛋白轻链3(LC3)的变化,以观察自噬情况;另外,用透射电镜观察rIL-12处理乳腺癌细胞前后自噬小体的变化。分别用胰岛素样生长因子1(IGF-1)激活磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)通路,再加入rIL-12处理后,蛋白免疫印迹法检测相关通路蛋白PI3K/Akt,哺乳动物雷帕霉素靶点(TOR)磷酸化变化及LC3的表达。结果与空白组相比,rIL-12组细胞的自噬标志蛋白LC3表达增高,差异有统计学意义(P<0.05),且增加程度呈时间和浓度依赖性;荧光显微镜观察自噬体形成的结果显示,与空白组相比,rIL-12处理组的细胞核周点状聚集的绿色荧光增多;使用电镜观察到rIL-12处理组明显有自噬小体形成。与rIL-12组相比,IGF-1+rIL-12组的LC3Ⅱ表达减少,差异有统计学意义(P<0.05)。结论rIL-12可以激活乳腺癌细胞自噬,并通过抑制PI3K/Akt信号通路的机制,促进自噬标志蛋白LC3,特别是LC3Ⅱ的表达。

Objective To investigate the effect of recombinant human IL-12(rIL-12)on breast cancer cell autophagy.Methods Two breast cancer cells(MDA-MB-231 and MCF7)were treated with rIL-12,and the changes of autophagy microtubule-associated protein light chain 3(LC3)were detected by immunoblotting and cell immunofluorescence.Observe autophagy;in addition,observe the change of autophagosomes in breast cancer cells before and after treatment with rIL-12 using transmission electron microscopy.Insulin-like growth factor 1(IGF-1)was used to activate the phosphatidylinositol-3 kinase/protein kinase B(PI3K/Akt)pathway,and then treated with rIL-12.Western blot was used to detect the related pathway protein PI3K/Akt,Mammalian rapamycin target(TOR)phosphorylation changes and LC3 expression.Results Compared with the blank group,the expression of autophagy marker protein LC3 in the cells of the rIL-12 group was significantly increased,the difference was statistically significant(P<0.05),and the degree of increase was time-and concentration-dependent;The results showed that,compared with the blank group,the green fluorescence around the nucleus in the rIL-12 treated group was significantly increased;it was observed that the autophagic bodies were formed in the rIL-12 treated group.Compared with rIL-12 group,the expression of LC3Ⅱin IGF-1+rIL-12 group was significantly reduced,the difference was statistically significant(P<0.05).Conclusion rIL-12 can activate the autophagy of breast cancer cells,and promote the expression of autophagy marker protein LC3,especially LC3Ⅱthrough the mechanism of inhibiting PI3K/Akt signaling pathway.