circNOX4通过靶向EZH2调控肝内胆管癌细胞增殖

CircNOX4 regulates the proliferation of intrahepatic cholangiocarcinoma cells by targeting EZH2

目的:探讨circNOX4通过靶向EZH2调控肝内胆管癌(ICC)细胞增殖的分子机制。方法:通过实时荧光定量聚合酶链反应(RT-qPCR)检测正常胆管上皮细胞(H69)和ICC细胞系(HCCC-9810、RBE、CCLP1、QBC939)中circNOX4的表达水平。荧光原位杂交技术(FISH)和核质分离实验确定circNOX4的亚细胞定位。EDU实验和细胞计数试剂盒(CCK-8)实验验证circNOX4对ICC细胞增殖的影响。蛋白质印迹法(Western blot)明确circNOX4与EZH2的相关性。两组间比较应用t检验,多组间比较应用单因素方差分析。结果:ICC细胞系circNOX4表达高于人正常胆管上皮细胞H69(H69:1.008±0.156,HCCC-9810:1.584±0.429,CCLP1:2.951±0.222,QBC939:6.404±0.511,RBE:10.305±2.988,F=23.97,P<0.05)。FISH实验和核质分离qRT-PCR实验可知,circNOX4主要分布于细胞核内。敲减circNOX4可以使RBE(2 d:siNC:0.646±0.119;sicircNOX4:0.358±0.032,t=4.67 P<0.05;3 d:siNC:1.041±0.052;sicircNOX4:0.620±0.084,t=8.54,P<0.05;4 d:siNC:1.707±0.070;sicircNOX4:1.007±0.081;t=13.07,P<0.05)和QBC939(2 d:siNC:0.534±0.010;sicircNOX4:0.403±0.017,t=13.52,P<0.05;3 d:siNC:0.887±0.034;sicircNOX4:0.589±0.023,t=14.68,P<0.05;4 d:siNC:1.496±0.151;sicircNOX4:0.865±0.122,t=6.51,P<0.05)细胞的增殖受到抑制。Western blot结果可知敲减circNOX4可以抑制EZH2的蛋白表达,敲减circNOX4同时回复EZH2可以恢复细胞增殖能力(4 d:siNC:1.611±0.061;sicircNOX4:0.810±0.025;sicircNOX4+EZH2:1.485±0.026;siNC比sicircNOX4:t=25.90,P<0.05;sicircNOX4比sicircNOX4+EZH2:t=37.56,P<0.05)。结论:circNOX4在ICC细胞系中高表达,并通过介导EZH2的表达调控细胞增殖能力。

Objective To explore the molecular mechanism of circNOX4 regulating the proliferation of intrahepatic cholangiocarcinoma cells by targeting the expression of EZH2.Methods The expression levels of circNOX4 in normal bile duct epithelial cells(H69)and intrahepatic cholangiocarcinoma cell lines(9810,RBE,CCLP1,QBC939)were detected by real-time quantitative polymerase chain reaction(RT-qPCR).The subcellular localization of circNOX4 was determined by fluorescence in situ hybridization(FISH)and nucleocytoplasmic separation experiments.The effect of circNOX4 on the proliferation of intrahepatic cholangiocarcinoma cells was verified by 5-Ethynyl-2’-deoxyuridine(EDU)and cell counting kit-8(CCK-8)experiments.The correlation between circNOX4 and EZH2 was verified by western blotting experiment.SPSS 24.0 software was used for statistical analysis,t-test was used for comparison between two groups,and one-way ANOVA was used for comparison between multiple groups.Results The expression of circNOX4 in intrahepatic cholangiocarcinoma cell lines were higher than that in human normal bile duct cell lines H69(H69:1.008±0.156,HCCC-9810:1.584±0.429,CCLP1:2.951±0.222,QBC939:6.404±0.511,RBE:10.305±2.988,F=23.97,P<0.05).FISH experiments and nuclear cytoplasmic separation QRT-PCR experiments showed that circNOX4 was mainly distributed in the nucleus.Knockdown circNOX4 inhibited the proliferation of RBE(2 d:siNC:0.646±0.119;sicircNOX4:0.358±0.032;t=4.67,P<0.05;3 d:siNC:1.041±0.052;sicircNOX4:0.620±0.084,t=8.54,P<0.05;4 d:siNC:1.707±0.070;sicircNOX4:1.007±0.081,t=13.07,P<0.05)and QBC939(2 d:siNC:0.534±0.010;sicircNOX4:0.403±0.017,t=13.52,P<0.05;3 d:siNC:0.887±0.034;sicircNOX4:0.589±0.023,t=14.68,P<0.05;4 d:siNC:1.496±0.151;sicircNOX4:0.865±0.122,t=6.51,P<0.05)cell lines.The Western blotting results showed that knockdown circNOX4 could inhibit the protein expression of EZH2 and cell proliferation(4 d:siNC:1.611±0.061;sicircNOX4:0.810±0.025;sicircNOX4+EZH2:1.485±0.026;siNC vs.sicircNOX4:t=25.90,P<0.05;sicircNOX4 vs.sicircNOX4+EZH2:t=37.56,P<0.05)which could be rescued by upregulating EZH2.Conclusion CircNOX4 is highly expressed in ICC cell lines and can regulate cell proliferation by mediating the expression of EZH2.