Rip2诱导人胰腺癌细胞自噬及其机制研究

目的:研究受体相互作用蛋白2(Rip2)是否诱导人胰腺癌细胞株Panc-1发生自噬及其作用机制。方法:用jet PRIME转染试剂将空质粒pEGFP-C2和重组质粒pEGFP-Rip2分别转染入Panc-1细胞,不做处理的细胞为对照组。转染后培养细胞48 h,采用Western blot检测Rip2、自噬相关蛋白[beclin-1和微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)]及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白的表达;透射电子显微镜观察自噬体的数量。结果:转染pEGFP-Rip2的细胞Rip2蛋白表达显著增加。与对照组和pEGFP-C2组相比,pEGFP-Rip2组细胞的beclin-1和LC3-Ⅱ蛋白表达水平显著升高(均P0.01);在透射电子显微镜下观察发现,pEGFP-Rip2组细胞内自噬体的数量明显多于对照组和pEGFP-C2组。pEGFP-Rip2组细胞的p-Akt和p-mTOR蛋白水平明显低于对照组和pEGFP-C2组(均P0.01),而总Akt和mTOR的蛋白水平变化不明显。结论:过表达Rip2可诱导Panc-1细胞发生自噬,其作用机制可能与Rip2抑制PI3K/Akt/mTOR通路的活化有关。

Rip2 induces autophagy and its mechanism in human pancreatic cancer cells

AIM:To explore whether receptor-interacting protein 2 (Rip2) induces autophagy and its under-lying mechanisms in human pancreatic cancer cell line Panc-1. METHODS:The empty plasmid pEGFP-C2 or recombinant plasmid pEGFP-Rip2 was transfected into the Panc-1 cells by jetPRIME reagent. The untreated cells served as control group. The protein levels of Rip2, autophagy-related molecules (beclin-1 and LC3-Ⅱ), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins were determined by Western blot 48 h after transfection. The morphological changes of the autophagosomes were observed by transmission electron microscopy. RESULTS:The protein level of Rip2 was significantly increased in the Panc-1 cells transfected with pEGFP-Rip2 plasmid. The protein expression of beclin-1 and LC3-Ⅱ in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group (all P<0.01). An increased number of autophagosomes was observed under transmission electron microscope in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group. Furthermore, the protein levels of p-mTOR and p-AKT in pEGFP-Rip2 group were lower than those in control group and pEGFP-C2 group (all P<0.01), while no significant difference of the total mTOR and AKT protein levels was found among the 3 groups. CONCLUSION:Rip2 induces autophagy in the Panc-1 cells and its mechanism may be related to inhibiting the activation of PI3K/Akt/mTOR pathway.