微小RNA-384通过调节己糖激酶2抑制人肝癌细胞的增殖

MicroRNA-384 inhibits the proliferation of human hepatocellular carcinoma cells through targeted regulation of the expression of hexokinase 2

目的探讨在人肝癌细胞中微小RNA(microRNA,miR)-384对己糖激酶2(HK2)的调节机制。方法应用荧光定量聚合酶链反应(PCR)的方法检测43例肝癌组织及癌旁组织中miR-384和HK2基因的表达水平,分为肝癌组织组及癌旁组织组;利用双荧光素酶报告基因实验验证miR-384与HK23’端非编码区(3’UTR)的具体结合位点,分为miR-384模拟物(mimics)组,空白对照(NC)组和HK2短发夹核糖核酸(shRNA)组;利用细胞计数试剂盒(CCK-8)实验观察miR-384和HK2对肝癌细胞HepG2(购自中国科学院上海细胞库)增殖能力的影响;采用挽救实验进一步验证HK2基因是否可以逆转miR-384对HepG2细胞增殖的调控作用。两样本均数间比较采用t检验。结果肝癌组miR-384表达水平低于癌旁组织组(0.42±0.07比1.11±0.06,t=18.210,P<0.01),差异有统计学意义,肝癌组HK2含量高于癌旁组织组(2.02±0.15比1.51±0.13,t=13.320,P<0.01),差异有统计学意义;miR-384 mimics与HK23’UTR-wt共转染组荧光素酶活性低于对照组(0.34±0.03比0.21±0.01,t=12.140,P<0.05),差异有统计学意义。而miR-384 mimics与HK2-3’UTR-mut共转染组荧光素酶活与对照组比较,差异无统计学意义(0.22±0.03比0.21±0.01,t=1.140,P>0.05)。转染miR-384 mimics组HK2的mRNA表达水平低于对照组(1.62±0.17比2.11±0.12,t=6.304,P<0.01),差异有统计学意义。CCK-8实验分析结果显示,miR-384 mimics组HepG2细胞的增殖能力低于对照组(0.82±0.07比1.31±0.11,t=2.941,P<0.01),差异有统计学意义。同样,HK2 shRNA组HepG2细胞的增殖能力也低于对照组(0.85±0.07比1.32±0.11,t=2.424,P<0.01),差异有统计学意义。miR-384 mimics和HK2过表达质粒共转染组HepG2细胞的增殖能力与NC组比较,差异无统计学意义(1.32±0.02比1.32±0.14,t=1.040,P>0.05)。结论miR-384可通过靶向调控HK2的表达,抑制HepG2细胞的增殖能力。

Objective To explore the regulation mechanism of microRNA-384(miR-384)on hexokinase 2(HK2)in human hepatocellular carcinoma.Methods:The fluorescence quantitative PCR was used to detect the expression level of miR-384 and HK2 in 43 cases of human hepatocellular carcinoma tissues and adjacent tissues,and they were divided into liver cancer tissue group and adjacent tissue group.Dual luciferase report assay was used to verify the specific binding site of miR-384 to HK23′UTR.The cell counting kit(CCK-8)was used to observe the effect of miR-384 and HK2 on the proliferation of liver cancer cells HepG2(purchased from Shanghai Cell Bank of Chinese Academy of Sciences),which were divided into miR-384 mimics group,NC group and HK2 shRNA group.Rescue experiments were used to further verify whether HK2 can reverse the proliferation effect of miR-384 on HepG2 cells,which were divided into miR-384 mimics and HK2 overexpression plasmid group and NC group.Statistical analysis was performed using SPSS 26.0 software,and the comparison between the two sample means was performed by t test.Results The miR-384 of the liver cancer group was lower than that of the adjacent tissue group(0.422±0.07 vs.1.109±0.06,t=18.21,P<0.01),and the HK2 of the liver cancer group was higher than that of the adjacent tissue group(2.016±0.15 vs.1.505±0.13,t=13.32),P<0.01).The luciferase activity of the miR-384 mimics and HK23′UTR-wt co-transfection group was significantly lower than that of the control group(0.34±0.03 vs.0.21±0.01,t=12.14,P<0.05).However,the luciferase activity of the miR-384 mimics co-transfected with HK2-3′UTR-mut was not statistically significant compared with the control group(0.22±0.03 vs.0.21±0.01,t=1.14,P>0.05).The mRNA expression level of HK2 in the miR-384 mimics group was significantly lower than that in the control group(1.621±0.17 vs.2.105±0.12,t=6.304,P<0.01).CCK8 experimental results showed that the proliferation ability of HepG2 cells in the miR-384 mimics group was significantly lower than that of the control group(0.817±0.07 vs.1.314±0.11,t=2.941,P<0.01).Similarly,the proliferation ability of HepG2 cells in the HK2 shRNA group was also sifnificantly lower than that of the control group(0.846±0.07 vs.1.322±0.11,t=2.424,P<0.01).Compared with the NC group,the proliferation ability of HepG2 cells in the miR-384 mimics and HK2 overexpression plasmid co-transfection group was not statistically significant(1.323±0.02 vs.1.318±0.14,t=1.04,P>0.05).Conclusion miR-384 can inhibit the proliferation of HepG2 cells via targeting HK2.