蛋白激酶B1基因介导内质网类似激酶/真核细胞翻译启始子2α信号通路参与肺癌细胞增殖及细胞凋亡进程的机制  

Mechanism of protein kinase B1 gene mediating protein kinase RNA like endoplasmic reticulum kinase/eukaryotic translation initiation factor 2 alpha signaling pathway in proliferation and apoptosis of lung cancer cells

作　　者：蔡仁中[1] 廖绪强[1] 黄明芳[1] 黎亮[1] Cai Renzhong;Liao Xuqiang;Huang Mingfang;Li Liang(Hainan Provincial People's Hospital(Hainan Hospital Affiliated to Hainan Medical College),Haikou 5703110,China)

机构地区：[1]海南省人民医院(海南医学院附属海南医院)胸外科,海口570311

出　　处：《中华实验外科杂志》2021年第4期684-688,共5页Chinese Journal of Experimental Surgery

摘　　要：目的探讨蛋白激酶B1(Akt1)基因介导内质网类似激酶(PERK)/真核细胞翻译启始子2α(eIF2α)信号通路参与肺癌细胞增殖及细胞凋亡的机制。方法选择人肺腺癌细胞系A549按实验转染方案随机分组,分为空白(Blank)组、沉默Akt1(si-Akt1)阴性对照(NC)组、si-Akt1组、过表达Akt1(OE-Akt1)NC组、OE-Akt1组、CCT020312(PERK选择性激动剂)组和OE-Akt1+CCT020312组。应用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)检测各组细胞中相关基因的mRNA和蛋白表达水平;同时分别应用噻唑蓝(MTT)法和流式细胞术检测各组细胞增殖和凋亡水平。两组间比较为t检验,多组间比较采用单因素方差分析。结果Si-Akt1组的Akt1的mRNA(0.99±0.05比0.45±0.03,t=16.04,P<0.05)和蛋白表达(0.32±0.03比0.12±0.01,t=10.95,P<0.05)低于si-Akt1 NC组。si-Akt1组PERK/p-PERK、eIF2α/p-eIF2α、GRP78和CHOP的mRNA(PERK:0.97±0.04比1.77±0.07,t=17.190,P<0.05;eIF2α:1.01±0.06比1.53±0.06,t=10.610,P<0.05;GRP78:1.06±0.03比1.98±0.08,t=18.650,P<0.05;CHOP:0.94±0.04比1.63±0.06,t=16.570,P<0.05)和蛋白表达(p-PERK:1.15±0.05比2.23±0.11,t=15.480,P<0.05;p-eIF2α:1.69±0.07比3.12±0.19,t=12.230,P<0.05;GRP78:0.84±0.05比2.87±0.15,t=22.650,P<0.05;CHOP:1.46±0.07比2.65±0.13,t=14.400,P<0.05)明显高于si-Akt1 NC组;细胞增殖能力明显低于si-Akt1 NC组(48 h:0.48±0.03比0.31±0.02,t=8.167,P<0.05;72 h:0.78±0.04比0.60±0.03,t=6.235,P<0.05),细胞凋亡率明显高于si-Akt1 NC组(8.58±1.34比23.4±2.4,t=9.250,P<0.05)。同时,CCT020312组PERK/p-PERK、eIF2α/p-eIF2α、GRP78和CHOP的mRNA(PERK:1.00±0.04比1.87±0.08,t=26.940,P<0.05;eIF2α:1.00±0.05比1.57±0.07,t=11.640,P<0.05;GRP78:1.00±0.03比2.02±0.10,t=41.640,P<0.05;CHOP:1.00±0.06比1.66±0.07,t=20.210,P<0.05)和蛋白表达(p-PERK:1.12±0.05比2.30±0.11,t=16.910,P<0.05;p-eIF2α:1.65±0.05比3.20±0.18,t=13.260,P<0.05;GRP78:0.80±0.04比2.74±0.15,t=21.640,P<0.05;CHOP:1.44±0.06比2.61±0.13,t=14.150,P<0.05)明显高�Objective To explore the role of protein kinase B1(also known as Akt1)gene mediating protein kinase RNA like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2 alpha(eIF2α)signaling pathway in the proliferation and apoptosis of lung cancer cells.Methods Human lung adenocarcinoma cell line A549 was selected to transfection for the construction of blank group,silencing Akt1(si-Akt1)negative control(NC)group,si-Akt1 group,overexpressing Akt1(OE-Akt1)NC group,OE-Akt1 group,CCT020312 group,and OE-Akt1+CCT020312 group.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the mRNA and protein expression levels of related genes.Meanwhile,3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay and flow cytometry were used to detect cell proliferation and apoptosis.T test was used to compare the two groups,and one-way ANOVA was used to compare multiple groups.Results Compared with si-Akt1 NC group,the expression of Akt1 mRNA(0.99±0.05 vs.0.45±0.03,t=16.04,P<0.05)and protein(0.32±0.03 vs.0.12±0.01,t=10.95,P<0.05)in si-Akt1 group was decreased,and si-Akt1 group showed increased mRNA(PERK:0.97±0.04 vs.1.77±0.07,t=17.190,P<0.05;eIF2α:1.01±0.06 vs.1.53±0.06,t=10.610,P<0.05;GRP78:1.06±0.03 vs.1.98±0.08,t=18.650,P<0.05;CHOP:0.94±0.04 vs.1.63±0.06,t=16.570,P<0.05)and protein expression(p-PERK:1.15±0.05 vs.2.23±0.11,t=15.480,P<0.05;p-eIF2α:1.69±0.07 vs.3.12±0.19,t=12.230,P<0.05;GRP78:0.84±0.05 vs.2.87±0.15,t=22.650,P<0.05;CHOP:1.46±0.07 vs.2.65±0.13,t=14.400,P<0.05)of PERK/p-PERK,eIF2α/p-eIF2α,GRP78 and CHOP,significantly decreased cell proliferation ability(48 h:0.48±0.03 vs.0.31±0.02,t=8.167,P<0.05;72 h:0.78±0.04 vs.0.60±0.03,t=6.235,P<0.05),and significantly increased apoptosis rate(8.58±1.34 vs.23.4±2.4,t=9.250,P<0.05).Meanwhile,CCT020312 group also indicated increased mRNA(PERK:1.00±0.04 vs.1.87±0.08,t=26.940,P<0.05;eIF2α:1.00±0.05 vs.1.57±0.07,t=11.640,P<0.05;GRP78:1.00±0.03 vs.2.02�

关 键 词：蛋白激酶B1 肺癌 增殖 凋亡 

分 类 号：R734.2[医药卫生—肿瘤]