环状RNA DLG1在肝细胞癌生物学中的作用  

Role of cyclic RNA DLG1 in the biology of hepatocellular carcinoma

作　　者：王谦 余伟 王涛 黄长山 黄涛 Wang Qian;Yu Wei;Wang Tao;Huang Changshan;Huang Tao(Department of Hepatobiliary and Pancreatic Surgery,the Affiliated Cancer Hospital of Zhengzhou University(Henan Cancer Hospital),Zhengzhou 450008,China)

机构地区：[1]郑州大学附属肿瘤医院(河南省肿瘤医院)肝胆胰外科,郑州450008

出　　处：《中华实验外科杂志》2023年第9期1749-1752,共4页Chinese Journal of Experimental Surgery

基　　金：河南省医学科技攻关计划(联合共建)(LHGJ20190658)。

摘　　要：目的探索环状RNA circDLG1在肝细胞癌(HCC)生物学进程中的作用。方法通过利用公共数据库分析circDLG1在肝细胞癌肿瘤组织及正常组织的表达水平,并利用定量实时聚合酶链反应(qRT-PCR)在细胞水平进行验证,qRT-PCR验证短发卡RNA(shRNA)转染效率,平板克隆实验分析circDLG1对HCC细胞增殖活力的影响。利用蛋白质印迹法(Western blot)和qRT-PCR检测circDLG1对HCC糖代谢相关葡萄糖的摄取、消耗、乳酸的产生和对索拉非尼耐药的影响。采用t检验或单因素方差分析组间差异。结果GSE97332公共数据库分析表明circDLG1在肿瘤组织中的表达高于正常组织组(8.632±0.861比4.833±0.357,t=6.482,P<0.05)。qRT-PCR结果表明circDLG1在肝细胞癌(SK-Hep-1、Huh7、HepG2、HepG3B和BEL7404组)中的表达量高于HCC空白对照组(3.358±0.325、8.297±0.557、5.624±0.571、4.028±0.534、6.488±0.386比1.566±0.136,t=6.228、8.372、7.864、6.486、6.318、7.571,P<0.05,P<0.01)。qRT-PCR验证转染shRNA可以使circRNA DLG1在HCC细胞中的表达水平低于HCC空白对照组(Hep3B:0.324±0.028、0.487±0.034比1.155±0.244,t=11.287、9.661,P<0.01;Huh7:0.283±0.016、0.425±0.024比1.117±0.486,t=11.498、10.087,P<0.01),平板克隆实验和生物化学实验结果表明敲低circRNA DLG1导致HCC细胞增殖低于HCC空白对照组(Hep3B:72.300±8.400、76.800±6.200比152.300±8.200,t=10.287、9.053,P<0.01;Huh7:46.500±4.100、52.500±7.800比173.800±9.600,t=20.384、16.343,P<0.01)、葡萄糖的摄取低于HCC空白对照组(Hep3B:138.942±10.133比98.323±7.922、68.669±7.924比109.355±7.654,t=12.035、14.546,P<0.01;Huh7:125.671±6.772比105.372±6.338、50.379±6.114比99.364±6.449,t=7.286、16.825,P<0.01)、细胞外酸化率低于HCC空白对照组(Hep3B:41.261±5.338比73.615±8.933,t=8.068,P<0.01;Huh7:44.871±8.245比69.348±9.742,t=10.323,P<0.01)及乳酸的产生低于HCC空白对照组(Hep3B:138.664±8.669比98.637±9.774、60.258±7.864比100.236±6.883,t=16.825、7.286,P<0Objective To explore the role of cyclic RNA circDLG1 in the biological process of hepatocellular carcinoma(HCC).Methods The expression levels of circDLG1 in HCC tissues and normal tissues adjacent to the cancer were analyzed by public databases and verified at the cellular level by quantitative real-time polymerase chain reaction(qRT-PCR).The short hairpin RNA(shRNA)transfection efficiency was verified by qRT-PCR,and plate cloning assay was used to analyze the effect of circDLG1 on the proliferation viability of HCC cells.The effects of circDLG1 on HCC cell glucose metabolism-related glucose uptake,lactate production and resistance to sorafenib were examined using Western blotting and qRT-PCR.Groups comparison was done using student-t test or ANOVA.Results GSE97332 public database analysis showed that circDLG1 expression level was elevated in tumor tissues(8.632±0.861 vs.4.833±0.357,t=6.482,P<0.05).The qRT-PCR results indicated that circDLG1 was highly expressed in HCC cells(3.358±0.325,8.297±0.557,5.624±0.571,4.028±0.534,6.488±0.386 vs.1.566±0.136,t=6.228,8.372,7.864,6.486,6.318,7.571,P<0.05,P<0.01).The qRT-PCR verified that transfection of shRNA reduced the expression level of circDLG1 in HCC cells(Hep3B vs.Blank:0.324±0.028,0.487±0.034 vs.1.155±0.244,t=11.287,9.661,P<0.01;Huh7 vs.Blank:0.283±0.016,0.425±0.024 vs.1.117±0.486,t=11.498,10.087,P<0.01).The results of plate cloning assay and biochemical experiments showed that circDLG1 could promote HCC cell proliferation(Hep3B vs.Blank:72.300±8.400,76.800±6.200 vs.152.300±8.200,t=10.287,9.053,P<0.01;Huh7 vs.Blank:46.500±4.100,52.500±7.800 vs.173.800±9.600,t=20.384,16.343,P<0.01,P<0.01),glucose uptake(Hep3B vs.Blank:138.942±10.133 vs.98.323±7.922,68.669±7.924 vs.109.355±7.654,t=12.035,14.546,P<0.01;Huh7 vs.Blank:125.671±6.772 vs.105.372±6.338,50.379±6.114 vs.99.364±6.449,t=7.286,16.825,P<0.01),and extracellular acidification rate(Hep3B vs.Blank:41.261±5.338 vs.73.615±8.933,t=8.068,P<0.01;Huh7 vs.Blank:44.871±8.245 vs.69.348±9.742,t=10

关 键 词：肝细胞癌 增殖 葡萄糖代谢 索拉非尼 

分 类 号：R735.7[医药卫生—肿瘤]