下调Nur77抑制PI3K/AKT信号通路促进肺腺癌细胞凋亡和自噬

Downregulation of Nur77 accelerates lung adenocarcinoma cells apoptosis and autophagy through the inhibition of the PI3K/AKT pathway

目的:探讨Nur77对肺腺癌细胞凋亡和自噬的影响及其调控机制。方法:收集右江民族医学院附属西南医院(百色市人民医院)经病理确诊的肺腺癌及癌旁组织5例,免疫荧光法检测Nur77的表达情况;生物信息学分析Nur77相关基因通路;体外培养人支气管上皮细胞BEAS-2B和人肺癌A549两株细胞系,分别记为BEAS-2B组和A549组。RT-qPCR检测两组细胞Nur77 mRNA的表达水平,Western blot检测Nur77、LC3、PI3K、p-PI3K、AKT、p-AKT的表达情况;免疫荧光检测Nur77的表达水平及亚细胞定位;将A549细胞分为si-NC组、si-Nur77组及Con组,si-NC组及si-Nur77组分别转染si-NC、si-Nur77,Con组未进行转染处理。RT-qPCR检测si-NC、si-Nur77组细胞Nur77 mRNA表达水平;Western blot检测Nur77、LC3、PI3K、p-PI3K、AKT、p-AKT、caspase-3、Bax、Bcl-2蛋白表达水平;流式细胞术检测细胞凋亡率。结果:肺腺癌组织Nur77蛋白表达水平高于癌旁组织;生物信息学分析显示Nur77与PI3K/AKT信号通路存在相关性;A549细胞Nur77 mRNA和蛋白表达水平均高于BEAS-2B细胞(P<0.001),A549细胞LC3Ⅱ/LC3Ⅰ比值、p-AKT/AKT比值均高于BEAS-2B细胞(P<0.05),A549细胞p-PI3K/PI3K比值高于BEAS-2B细胞(P<0.01);si-Nur77组Bcl-2表达低于si-NC组(P<0.05),si-Nur77组Caspase-3、Bax、LC3Ⅱ/LC3Ⅰ比值高于si-NC组(P<0.01),而p-AKT/AK、p-PI3K/PI3K在si-Nur77组低于si-NC组(P<0.05),si-Nur77组细胞凋亡率高于si-NC组(P<0.05)。结论:下调Nur77可促进肺腺癌细胞自噬和凋亡,其作用机制可能与抑制PI3K/AKT信号通路有关。

Objective:To investigate the role and mechanism of Nur77 in lung adenocarcinoma cell apoptosis and autophagy.Methods:Five cases of lung adenocarcinoma tissues which were confirmed by pathology and para-cancerous tissues were collected from the Affiliated Southwest Hospital of Youjiang Medical University for Nationalities(People′s Hospital of Baise).The expression of Nur77 in lung adenocarcinoma and para-cancerous tissues was detected with immunofluorescence.Nur77-associated pathways were analyzed with bioinformatics analysis.Human bronchial epithelial(BEAS-2B)cells and human lung cancer(A549)cells were cultured in vitro and designated as BEAS-2B and A549 groups,respectively.In both groups,Nur77 mRNA expression was measured with RT-qPCR,the expression of Nur77,LC3,PI3K,p-PI3K,AKT,and p-AKT was assessed with western blotting,and the fluorescence intensity of Nur77 was determined with immunofluorescence.A549 cells were transfected with si-NC or si-Nur77,referred to as si-NC or si-Nur77 groups,respectively,and non-transfected cells serving as the control group(Con).Nur77 expression was analyzed with RT-qPCR,and the expression of Nur77,LC3,PI3K,p-PI3K,AKT,p-AKT,caspase-3,Bax,and Bcl-2 was examined using Western blot.Cell apoptosis was assessed by flow cytometry.Results:Lung adenocarcinoma tissues exhibited higher levels of Nur77 protein expression compared to para-cancerous tissues.Bioinformatics analysis demonstrated a correlation between Nur77 and the PI3K/AKT pathway.The mRNA and protein expression of Nur77(P<0.001)and the ratio of LC3Ⅱ/LC3Ⅰ,p-AKT/AKT(P<0.05),and p-PI3K/PI3K(P<0.01)were higher in the A549 group than in the BEAS-2B group.Compared with the si-NC group,the si-Nur77 group exhibited decreased Bcl-2 expression(P<0.05),increased Caspase-3 and Bax expression(P<0.01),and elevated LC3Ⅱ/LC3Ⅰratio(P<0.01),along with reduced p-AKT/AKT(P<0.05)and p-PI3K/PI3K(P<0.01)ratios and significantly enhanced apoptosis(P<0.05).Conclusion:Nur77 downregulation accelerates autophagy and apoptosis in lung cancer cells,which may be achieved by the inhibition of the PI3K/AKT pathway.