摘要
目的:探讨NOD8(也称NLRP10或PYNOD)是否能抑制胰腺癌细胞发生自噬及其可能机制,并研究凋亡对NOD8调控自噬的影响。方法:用JetPRIME阳离子聚合物将空质粒pEGFP-C2和重组质粒pEGFP-NOD8分别转染入胰腺癌Panc-1细胞,并设立空白对照组,48 h后应用Western blot检测NOD8蛋白、自噬相关蛋白(beclin-1和LC3-Ⅱ)以及磷脂酰肌醇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(Akt、p-Akt、mTOR和p-mTOR)的表达;并采用免疫荧光染色法观察细胞LC3斑点的数量。此外,用caspase抑制剂Z-VAD-FMK作用于过表达NOD8的Panc-1细胞后,Western blot检测beclin-1和LC3-Ⅱ蛋白的表达水平;免疫荧光染色法观察细胞LC3斑点数量。结果:pEGFP-NOD8组细胞NOD8蛋白表达明显高于对照组和pEGFP-C2组(P<0.01)。与对照组和pEGFP-C2组相比,pEGFP-NOD8组细胞beclin-1和LC3-Ⅱ蛋白表达及细胞LC3斑点数量显著降低;并且pEGFP-NOD8组细胞p-Akt及p-mTOR蛋白表达量显著上升(P<0.01),而AKT和mTOR蛋白表达无明显差异。与pEGFP-NOD8组相比,pEGFP-NOD8+Z-VAD-FMK组细胞beclin-1和LC3-Ⅱ蛋白表达水平及LC3斑点数量显著高于pEGFP-NOD8组。结论:NOD8可抑制Panc-1细胞自噬,其机制可能与激活PI3K/Akt/mTOR通路有关;凋亡可增强NOD8对自噬的抑制作用。
AIM:To explore whether NOD8 inhibits autophagy in human pancreatic cancer cells and its underlying mechanisms,and to investigate the effect of apoptosis on the autophagy regulated by NOD8.METHODS:The empty plasmid pEGFP-C2 and recombinant plasmid pEGFP-NOD8 were transfected into the Panc-1 cells using JetPRIME reagent.The untransfected cells served as control group.The protein levels of NOD8,autophagy-related proteins beclin-1 and LC3-II,and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway-related proteins Akt,p-Akt,mTOR and p-mTOR were determined by Western blot 48 h after transfection.Meanwhile,the number of LC3 spots was quantified by immunofluorescence staining.Furthermore,after a broad caspase inhibitor Z-VAD-FMK was applied to NOD8-over-expressing cells,the protein expression levels of beclin-1 and LC3-II were detected by Western blot and the number of LC3 spots was observed by immunofluorescence staining.RESULTS:The protein level of NOD8 in pEGFP-NOD8 group was significantly higher than that in control group and pEGFP-C2 group(P<0.01).The protein expression of beclin-1 and LC3-II,and the number of LC3 spots in pEGFP-NOD8 group were significantly decreased as compared with control group and pEGFP-C2 group.Moreover,the protein levels of p-AKT and p-mTOR in pEGFP-NOD8 group were higher than those in control group and pEGFP-C2 group,while no significant difference of mTOR and AKT protein expression was found among these 3 groups.Furthermore,the protein levels of beclin-1 and LC3-II,and the number of LC3 spots in pEGFP-NOD8+Z-VAD-FMK group were significantly increased compared with pEGFP-NOD8 group.CONCLUSION:NOD8 inhibits autophagy in the Panc-1 cells and its mechanism may be related to the activation of PI3K/Akt/mTOR pathways.Apoptosis enhances the inhibitory effect of NOD8 on autophagy.
作者
梁若龙
曾琪
王晗月
胡巢凤
罗森
LIANG Ruo-long;ZENG Qi;WANG Han-yue;HU Chao-feng;LUO Sen(Department of Pathophysiology,Key Laboratory of Pathophysiology,State Administration of Traditional Chinese Medicine of The People’s Republic of China,Basic Medical School of Jinan University,Guangzhou 510632,China;The First Affiliated Hospital of Gannan Medical University,Ganzhou 341000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2019年第12期2215-2220,共6页
Chinese Journal of Pathophysiology
基金
广东省自然科学基金资助项目(No.S2012010008161)
