miR-612对肝细胞癌干细胞特性影响及机制研究

Mechanism of miR-612 on hepatocellular carcinoma stem cell characteristics

目的:探讨miR-612对肝细胞癌(HCC)干细胞特性的影响并探讨其机制。方法:采用实时定量PCR(qRT-PCR)法检测miR-612在HCC细胞系和正常肝细胞系中的表达;用脂质体将miR-612 mimic/对照转染至HepG2细胞中,采用细胞肿瘤球形成和软琼脂克隆形成实验检测miR-612对HepG2细胞干性的影响;qRT-PCR及蛋白免疫印迹试验(Western blot)检测HepG2细胞中CD133、Nanog的mRNA和蛋白表达水平。用荧光素酶报告基因试验检测miR-612对Nanog基因的直接调控作用。结果:qRT-PCR结果显示miR-612在LM3、HepG2、Hep3B HCC细胞中相对表达量分别为2.00±0.10、1.01±0.06、1.51±0.05,显著低于在肝正常细胞LO2中的相对表达量5.23±0.43(P值均<0.05);HepG2转染miR-612 mimic后,细胞悬浮肿瘤球形成和软琼脂克隆球形成的数目均显著减少(P值均<0.05),细胞中CD133、Nanog的mRNA和蛋白表达均减少(P值均<0.05)。双荧光素酶报道基因结果显示miR-612可以特异性直接靶向作用于Nanog基因。结论:在HCC HepG2细胞中,高表达miR-612能够抑制细胞干性表达,可能是通过下调Nanog基因表达实现的。

Objective:To investigate the effect of miR-612 on the characteristics of hepatocellular carcinoma(HCC)stem cells and explore its mechanism.Methods:Real-time PCR(qRT-PCR)was used to detect the expression of miR-612 in HCC cell lines and normal hepatic cell lines;miR-612 mimic/control were transfected into HepG2 cells by liposome,cell tumor sphere formation and soft agar colony formation assay was used to detect the effect of miR-612 on the stemness of HepG2 cells;qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of CD133 and Nanog in HepG2 cells;luciferase reporter assay detect the direct regulation of miR-612 on the Nanog gene.Results:The qRT-PCR results showed that the relative expression levels of miR-612 in LM3,HepG2 and Hep3 B HCC cells were 2.00±0.10,1.01±0.06 and 1.51±0.05,respectively,which were significantly lower than those in normal liver cells(5.23±0.43,P<0.05).After HepG2 transfection with miR-612 mimic,the number of cell suspension tumor sphere formation and soft agar cloned sphere formation were significantly decreased(P<0.05).The mRNA and protein expression of CD133 and Nanog were decreased in the cells(P<0.05);the dual luciferase reporter gene results showed that miR-612 could specifically target the Nanog gene directly.Conclusion:The high expression of miR-612 in HCC HepG2 cells can inhibit the stemness of the cancer cells,which may be achieved by down-regulating the expression of Nanog gene.