长链非编码RNA HCG22调控蛋白激酶B/P70S6激酶通路对胃癌细胞生物学行为的影响  

Effects of long non-coding RNA HCG22 on the biological behavior of gastric cancer cells by regulating protein kinase B/P70S6 kinase pathway

作　　者：张牙 张卓奇[1] 问明[1] 郭剑[1] 冯长平 彭鑫宇[1] 李杰[1] 张爱民[1] Zhang Ya;Zhang Zhuoqi;Wen Ming;Guo Jian;Feng Changping;Peng Xinyu;Li Jie;Zhang Aimin(Department of Gastrointestinal Surgery,Affiliated Hospital of Hebei University,Baoding 071000,China)

机构地区：[1]河北大学附属医院胃肠外科,保定071000

出　　处：《中华实验外科杂志》2022年第6期1102-1105,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨长链非编码RNA HCG22(lncRNA HCG22)对胃癌细胞生物学行为的影响及其机制。方法实时定量聚合酶链反应(RT-qPCR)检测正常胃上皮细胞GES-1和胃癌MGC-803、SGC-7901细胞lncRNA HCG22的表达;采用慢病毒过表达和对照载体感染MGC-803细胞, 分为过表达组(LV-HCG22-OE)、对照组(LV-NC)和空白组(Blank);细胞计数试剂盒(CCK-8)法检测24、48、72 h的吸光度值, 集落形成实验检测细胞集落形成率;划痕愈合实验检测迁移能力;流式细胞术分析周期和凋亡;免疫荧光观察半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)荧光;蛋白质印迹法(Western blot)检测凋亡蛋白、自噬标志物以及蛋白激酶B(Akt)/P70S6激酶(P70S6K)通路蛋白的变化。两组间比较采用t检验。结果 lncRNA HCG22在MGC-803(0.026±0.011)和SGC-7901的表达(0.048±0.012)低于GES-1细胞(1.000±0.123, t=7.871、7.694, P<0.01)。上调lncRNA HCG22后, 抑制细胞的增殖能力, LV-HCG22-OE组的吸光度值低于LV-NC组(24 h为0.295±0.015比0.501±0.014, 48 h为0.526±0.012比0.952±0.048, 72 h为0.929±0.016比1.381±0.020, t=10.170、8.614、17.800, P<0.01);集落形成率[(36.250±2.000)%比(53.880±3.375)%, t=4.493, P<0.05]和迁移能力均低于LV-NC组[24 h为(32.940±1.315)%比(50.220±1.120)%, 36 h为(63.970±1.500)%比(79.650±1.000)%, t=10.010、8.698, P<0.05]。LV-HCG22-OE组G2/M期细胞比例增加, G1期减少;细胞凋亡率增加(17.980±3.120比3.667±0.997, t=4.372, P<0.05)。共聚焦显微镜观察到Caspase-3荧光信号增加, Western blot结果显示, LV-HCG22-OE组B细胞淋巴瘤因子-xL(bcl-xL)和p62蛋白水平低于LV-NC组(t=7.686、5.376, P<0.05), B细胞淋巴瘤因子-2相关X蛋白(bax), 微管相关蛋白1轻链3B(LC3-Ⅱ)蛋白水平高于LV-NC组(t=9.311、4.768, P<0.05);LV-HCG22-OE组磷酸化蛋白激酶B(p-Akt)、磷酸化P70S6激酶(p-P70S6K)蛋白水平低于LV-NC组(p-Akt/Akt为0.426±0.085比0.891±0.012, t=5.395, P<0.05;p-P70S6K/P70S6K为0.605±0.019比1.027±0.072, t=5Objective To investigate the effects and mechanism of long non-coding RNA HCG22(lncRNA HCG22)on proliferation,migration,cycle,apoptosis and autophagy of gastric cancer cells.Methods Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of lncRNA HCG22 in normal gastric epithelial cells GES-1 and gastric cancer cells(MGC-803 and SGC-7901).The MGC-803 cells were infected using lentiviral overexpression and a control vector,and cells were divided into overexpression group(LV-HCG22-OE),control group(LV-NC)and blank group(blank).The absorbance was measured at 24,72 and 42 h by cell counting kit-8(CCK-8)assay,and the cell activity was determined by colony formation assay.The migration ability was explored by wound assay.Cell cycle percentage and apoptosis rate were detected by flow cytometry.The fluorescence of cysteinyl aspartate-specific protease(Caspase)-3 was observed by immunofluorescence.Western blotting was used to detect the expression of apoptosis proteins,autophagy markers and protein kinase B(Akt)/P70S6 kinase(P70S6K)pathway.T-test was used for comparison between groups.Results The expression levels of lncRNA HCG22 in MGC-803(0.026±0.011)and SGC-7901(0.048±0.012)were lower than in GES-1(1.000±0.123,t=7.871,7.694,P<0.01).Up-regulation of lncRNA HCG22 inhibited cell proliferation.The absorbance in LV-HCG22-OE group was lower than in LV-NC group(24 h=0.295±0.015 vs.0.501±0.014,48 h=0.526±0.012 vs.0.952±0.048,72 h=0.929±0.016 vs.1.381±0.020,t=10.170,8.614,17.800,P<0.01).The colony formation rate[(36.250±2.000)%vs.(53.880±3.375)%,t=4.493,P<0.05]and migration ability[24 h=(32.940±1.315)%vs.(50.220±1.120)%,36 h=(63.970±1.500)%vs.(79.650±1.000)%,t=10.010,8.698,P<0.05)]were lower than those in LV-NC group.The percentage in G2/M phase,apoptosis rate(17.980±3.120 vs.3.667±0.997,t=4.372,P<0.05)and Caspase-3 fluorescence were increased in LV-HCG22-OE group.Western blotting results showed that the protein expression of B cell lymphoma/leukemia-xL(bcl-xL)and p62 was low

关 键 词：胃癌 长链非编码RNA 生物学行为 蛋白激酶B/P70S6激酶 

分 类 号：R735.2[医药卫生—肿瘤]