miR-150、CCNB1及MFN2参与调控肝癌细胞Huh-7凋亡并抑制其侵袭迁移的机制研究

Mechanism of miR-150, CCNB1 and MFN2 involved in regulating apoptosis of Huh-7 cells and inhibiting invasion and migration

目的:探究miR-150、细胞周期蛋白B1(CCNB1)及线粒体融合蛋白2(MFN2)参与调控肝癌细胞Huh-7凋亡并抑制其侵袭迁移的机制。方法:将肝癌细胞Huh-7分为对照组(control组)、阴性对照组(NC组)和miR-150过表达组(mimic组),通过质粒转染构建miR-150过表达细胞系。CCK-8和流式细胞术应用于检测细胞活力的凋亡,细胞划痕实验和Transwell应用于检测迁移和侵袭,qPCR检测miRNA和mRNA水平,WB检测蛋白的相对表达水平。结果:miR-150可显著抑制Huh-7的细胞活力而促进凋亡(P<0.01)。培养24 h后mimic组迁移率、侵袭率均显著低于control组和NC组(P<0.01)。Mimic组的CCNB1蛋白和mRNA的表达水平显著低于control组和NC组(P<0.01),MFN2显著高于control组和NC组(P<0.01)。结论:在肝癌细胞Huh-7中,miR-150可能通过下调CCNB1或上调MFN-2的水平抑制肝癌细胞的增殖、迁移、侵袭,并促进其凋亡。

Objective:To explore miR-150,cyclin B1(CCNB1)and mitochondria-associated protein 2(MFN2)are involved in the regulation of apoptosis and inhibition of invasion and migration of hepatoma cells Huh-7.Methods:Hepatoma cells Huh-7were divided into control group,negative control group(NC)and miR-150overexpression group(mimic).The miR-150 overexpressing cell line was constructed by plasmid transfection.CCK-8and flow cytometry were used to detect apoptosis in cell viability.Cell scratch assays and Transwell were applied to migration and invasion,qPCR to detect miRNA and mRNA levels, and WB to detect relative expression levels of proteins.Results:miR-150significantly inhibited the cell viability of Huh-7 and promoted apoptosis(P<0.01).After 24hof culture,the mobility of control group and NC group were (83.54±4.66)% and(85.57±4.74)%,respectively,and mimic group was(49.63±3.78)%,which was significantly lower than control group and NC group(P<0.01).After 24hof culture,the invasive rate of control group and NC group were(100.56±2.87)%and (101.63±3.74)%,respectively,and mimic group was(51.63±5.32)%,which was significantly lower than control group and NC group(P<0.01).The expression levels of CCNB1protein and mRNA in Mimic group were significantly lower than thosin control group and NC group(P<0.01),and MFN2was significantly higher than control group and NC group(P<0.01). Conclusion:In hepatoma cell Huh-7,miR-150may inhibit the proliferation,migration,invasion and apoptosis of hepatoma cells by regulating CCNB1or up-regulating MFN-2levels.