miR-486-5p通过负向调控AMPK/Sirt1信号通路参与肝细胞性肝癌细胞增殖

miR-486-5p participates in hepatocellular carcinoma cell proliferation by negatively regulating AMPK/Sirt1 signaling pathway

目的:探究miR-486-5p在肝细胞性肝癌(HCC)中的变化及其调节HCC细胞系增殖的可能机制。方法:q-PCR法检测98例健康人血浆和87例HCC患者组织与血浆miR-486-5p含量,并采用Pearson相关性分析其与甲胎蛋白(AFP)升高的相关性,同时检测人HCC癌细胞系SMMC-7721、Bel-7402、MHCC97、HepG2、Hep3B、Huh-7、MS751和正常肝细胞LO2细胞及转染0,25,50,100μmol/L miR-486-5p类似物(miR-486-5p mimic) 24 h后LO2、MHCC97、HepG2和Huh-7细胞的miR-486-5p含量;CCK-8法检测转染miR-486-5p类似物2,12,24,48,72 h的LO2、MHCC97、HepG2和Huh-7细胞的增殖率;Western Blot检测Cyclin D1、Cyclin E1、CDK4、CDK6、p-AMPK、AMPK和Sirt1蛋白表达量;qPCR检测转染CCND1、CCNE、CDK4、CDK6 mRNA表达量。结果:HCC患者癌组织miR-486-5p含量明显低于癌旁组织,血浆中miR-486-5p含量明显低于正常人;HCC患者癌组织和血浆中miR-486-5p含量与血浆AFP含量呈显著负相关性,相关系数分别为-0. 1554和-0. 2228(P<0. 001);除Huh-7细胞外,人HCC癌细胞系SMMC-7721、Bel-7402、MHCC97、HepG2、Hep3B和MS751细胞中miR-486-5p含量明显低于LO2细胞(P<0. 05);相比于阴性对照类似物(NC)处理组,0至100μmol/L的miR-486-5p类似物处理LO2和Huh-7细胞24 h组的miR-486-5p水平均没有明显的变化(P>0. 05),50μmol/L和100μmol/L的miR-486-5p类似物处理24 h的MHCC97和HepG2细胞,相比于NC处理组,其miR-486-5p水平均显著增加(P<0. 05);miR-486-5p类似物处理LO2组的增殖率相比于NC和Control组无明显的变化。相比于NC和Control组,miR-486-5p类似物处理HepG2和MHCC97细胞24 h的增殖率相比于2 h的显著降低,分别为(77. 70±7. 53)%和(86. 61±1. 08)%。且具有时间依赖性。而miR-486-5p类似物处理Huh-7细胞48 h组增殖率为(84. 79±2. 33)%,明显低于2 h组;miR-486-5p类似物转染HepG2和MHCC97细胞24 h的Cyclin D1、Cyclin E、CDK6蛋白与mRNA表达量明显低于NC组(P<0. 05),HepG2的miR-486-5p类似物处理CDK4相比于NC组无明显变化(P>0. 05),MHCC97的miR-486-5p类似物组CDK4相比于NC组明显降低(P<0. 05)。p-AMPK与Sirt1蛋白水平均明显低于NC组。结论:过表达miR-486-5p通过下调p-AMPK/Sirt1通路阻断HCC肿瘤细胞细胞周期,并抑制癌细胞增殖。

Objective:To explore the changes of microRNA-486-5 p in hepatocellular carcinoma(HCC)and the potential mechanism of regulating the proliferation of HCC cell.Methods:q-PCR was used to detect the levels of miR-486-5 p in 98 healthy subjects and 87 patients with HCC.Pearson correlation was used to analyze the correlation between miR-486-5 p and AFP.MiR-486-5 p level in human HCC cancer cell lines SMMCMC-7721,Bel-7402,MHCC97,MHCC97,HepG2,Hep3 B,Huh-7,MS751 and normal hepatocyte LO2 cells were detected by q-PCR,also done in transfected LO2,MHCC97,MHCC97,MHCC97,HepG2 cells;the proliferation rate of LO2,MHCC97,HepG2 and Huh-7 cells after treated by miR-486-5 p analogues at the end of 2 h,12 h,24 h,48 h and 72 h post-treatment were detected by CCK-8 method;Cyclin D1,Cyclin E1,CDK4,CDK6,p-AMPK,AMPK and Sirt 1 contents were detected by Western Blot;and mRNA levels of CCN1,CCNE,CDK4 and CDK6 were detected by q-PCR.Results:MiR-486-5 p content in HCC patients was significantly lower than that in peritumoral tissues,and plasma miR-486-5 p was significantly lower than that of normal persons;Cancer tissue and plasma miR-486-5 p of HCC patients were negatively correlated with plasma AFP content,and the correlation coefficients were-0.155 4 and-0.222 8(both P<0.001),respectively;except for Huh-7 cells,miR-486-5 p content in human HCC cancer cell lines SMMCMC-7721,Bel-7402,MHCC97,MHCC97,HepG2,Hep3 B,Huh-7,and MS75 were higher than those in normal hepatocyte LO2 cells;compared with each NC group,miR-486-5 p content in MHCC97 and HepG2 were higher after 50μmol/L and 100μmol/L analogues treatment for 24 h(P<0.05),but no significant changes were found in the treatment group of LO2 and Huh-7 cell lines(P>0.05).The proliferation rate of LO2 transfected with 50μmol/L miR-486-5 p analogue for 24 h had no significant change as compared with that in NC and Control groups;the proliferation rates of HepG2 and MHCC97 at 24 h was(77.70±7.53)%and(86.61±1.08)%,respectively after treatment with miR-486-5 p analogues,which were significantly lower than those at 2 hours in time-dependent manner.The proliferation rate of Huh-7 cells after treatment with miR-486-5 p analogue was(84.79±2.33)%at 48 h,which was significantly lower than that of 2 h group(P<0.05).Protein and mRNA levels of p-AMPK,Sirt1,Cyclin D1,Cyclin E and CDK6 in HepG2 and MHCC97 cells transfected with miR-486-5 p analogue were significantly lower than those in NC group at 24 h(P<0.05),but no significant changes in CDK4 were found(P>0.05).Conclusion:Overexpression of miR-486-5 p can inhibit the proliferation of cancer cells by blocking the cell cycle through down-regulating p-AMPK/Sirt1 pathway.