鳖甲煎丸含药血清通过miR-140调控肝癌干细胞增殖能力及干性的机制研究

Study on the Mechanism of Regulating the Proliferation Ability and Dryness of Hepatocellular Carcinoma Stem Cells Through miR-140 in Biejiajian Pills(鳖甲煎丸)

目的 研究miR-140在肝癌干细胞(liver cancer stem cells,LCSCs)中的生物学功能,以及鳖甲煎丸含药血清抑制肝癌干细胞特性的干预效应。方法 以人肝癌细胞株(Huh7)为研究对象,无血清培养基分选法分选LCSCs,用流式细胞仪鉴定富集的LCSCs,并转染miR-140慢病毒。分为Cin组、Control组、BJJP组、miR-140 mimics NC组、miR-140 mimics组、BJJP+miR-140 inhibitor组。用CCK-8法测定细胞增殖能力,RTPCR检测miR-140、CD133、CD24、EpCAM、AFP表达,Western blot法检测CD133、CD144、EpCAM、AFP蛋白表达。结果 检测显示Con组与Control组细胞增殖能力、CD133、CD24、EpCAM、AFP的蛋白及RNA表达无统计学意义(P>0.05);与miR-140 mimics NC组相比,miR-140 mimics组的肝癌干细胞的增殖能力、CD133、CD24、EpCAM、AFP的RNA及蛋白表达水平均明显减少(P<0.01),但miR-140表达明显增强(P<0.05);与Control组相比,BJJP组和BJJP+miR-140 inhibitor组的细胞增殖能力、CD133、CD24、EpCAM、AFP的RNA及蛋白表达水平显著降低(P<0.01),且BJJP组的细胞增殖能力、CD133、CD24、EpCAM、AFP的RNA及蛋白表达水平低于BJJP+miR-140inhibitor组(均P<0.05)。结论 鳖甲煎丸能抑制LCSCs的增殖能力及干性,其机制可能通过上调miR-140的表达发挥作用。

Objective To study the biological function of miR-140 in liver cancer stem cells(LCSCs)and the intervention effect of Biejiajian Pills(鳖甲煎丸)containing serum on the inhibition of liver cancer stem cells.Methods Human hepatocellular carcinoma cell line(Huh7)was selected as the research object,LCSCs were sorted by serum-free medium sorting method,and the enriched LCSCs were identified and transfected with miR-140 lentivirus by flow cytometry.They were divided into Cin group,Control group,BJJP group,miR-140 mimics NC group,miR-140 mimics group and BJJP+miR-140 inhibitor group.The cell proliferation capacity was determined by CCK-8 method,the expressions of miR-140,CD133,CD24,EpCAM and AFP were detected by RT-PCR,and the expressions of CD133,CD144,EpCAM and AFP were detected by Western blot.Results There was no significant differences in cell proliferation capacity,protein and RNA expressions of CD133,CD24,EpCAM and AFP between Con group and Control group(P>0.05).Compared with the miR-140 mimics NC group,the proliferation ability,RNA and protein expression levels of CD133,CD24,EpCAM and AFP of HCC stem cells in the miR-140 mimics group were significantly decreased(P<0.01),but the expression of miR-140 was significantly increased(P<0.05).Compared with Control group,cell proliferation capacity,RNA and protein expression levels of CD133,CD24,EpCAM and AFP were significantly decreased in BJJP group and BJJP+miR-140 inhibitor group(P<0.01).Moreover,cell proliferation capacity,RNA and protein expression levels of CD133,CD24,EpCAM and AFP in BJJP group were lower than those in BJJP+miR-140 inhibitor group(all P<0.05).Conclusion Biejiajian Pills can inhibit the proliferation and dryness of LCSCs,and its mechanism may be through up-regulation of miR-140 expression.