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目的:探究miR-146a/b在Hep3B的表达及其上游调控机制和下游靶标蛋白。方法:MTT法检测人正常肝细胞株LO2和人肝癌细胞株HepG2、Hep3B的增殖活性。分别提取3个细胞的总RNA和蛋白,应用RT-qPCR和蛋白印记法分别检测细胞株中miR-146a/b的表达和细胞外调节激酶1/2(ERK)、磷酸化ERK 1/2(p-ERK1/2)、磷酸化蛋白激酶B(p-AKT)、NF-κB抑制蛋白α亚基(IκBα)、白介素1受体关联激酶1(IRAK1)、肿瘤坏死因子受体相关蛋白6(TRAF6)的蛋白水平。用抑制剂分别抑制Hep3B细胞PI3K、AKT、ERK、NF-κB信号分子的活性并检测miR-146a/b的表达水平及IRAK1、TRAF6的蛋白水平。结果 Hep3B细胞增殖活力和miR-146a/b表达水平显著高于LO2和HepG2(P<0.01)。蛋白印记结果显示,以LO2为对照组,Hep3B细胞的pERK1、ERK1、p-AKT、IκB的蛋白水平明显升高,分别为LO2的10.87、24.68、6.67和1.92倍;IRAK1、TRAF6的蛋白水平明显降低,分别为LO2的0.23和0.003倍。与LO2细胞相比,HepG2细胞的IκB和IRAK1蛋白表达明显升高,分别为LO2的4.46和2.69倍。抑制Hep3B细胞的PI3K和AKT活性12 h和24 h,miR-146a和miR-146b的表达水平显著降低(P<0.05);抑制ERK和NF-κB的活性12 h和24 h,miR-146a/b的表达水平没有显著变化。抑制PI3K、AKT、ERK、NF-κB信号通路活性均可上调TRAF6和下调IRAK1的蛋白表达水平。结论:在恶化程度较高的Hep3B细胞中,PI3K/AKT可通过上调miR-146a/b的表达进而下调TRAF6的蛋白水平,这一机制为肝肿瘤发生发展的机理研究提供了重要线索。
Objective: To investigate the expression of miR-146a / b in Hep3B and its upstream regulatory mechanism and downstream target proteins. Methods: The proliferation activity of human normal liver cell line LO2 and human hepatocellular carcinoma cell lines HepG2 and Hep3B were detected by MTT assay. The total RNA and protein of three cells were extracted respectively. The expression of miR-146a / b and ERK, ERK 1/2 (phosphorylated ERK 1/2) in cell lines were detected by RT-qPCR and Western blot respectively p-ERK1 / 2, p-AKT, IκBα, IRAK1, TNFR6 TRAF6) protein levels. Inhibitory activities of PI3K, AKT, ERK and NF-κB in Hep3B cells were detected by enzyme-linked immunosorbent assay (ELISA). The expression of miR-146a / b and the protein levels of IRAK1 and TRAF6 were detected. Results The proliferation activity and the expression of miR-146a / b in Hep3B cells were significantly higher than those in LO2 and HepG2 cells (P <0.01). The results of Western blotting showed that the protein levels of pERK1, ERK1, p-AKT and IκB in Hep3B cells were significantly increased with LO2 as control group, which were 10.87, 24.68, 6.67 and 1.92 times higher than that of LO2, respectively. The protein levels of IRAK1 and TRAF6 were significantly Decreased, respectively, 0.23 and 0.003 times LO2. Compared with LO2 cells, the expressions of IκB and IRAK1 in HepG2 cells were significantly increased, which were 4.46 and 2.69 times of LO2, respectively. Inhibition of PI3K and AKT activity in Hep3B cells decreased the expression of miR-146a and miR-146b at 12 h and 24 h (P <0.05), inhibited the activity of ERK and NF-κB at 12 h and 24 h, The expression level of b did not change significantly. Inhibition of PI3K, AKT, ERK, NF-κB signaling pathway activity can upregulate TRAF6 and downregulate the protein expression of IRAK1. Conclusion: PI3K / AKT down-regulates the expression of miR-146a / b in Hep3B cells with a higher degree of exacerbation, thereby down-regulating the protein level of TRAF6. This mechanism provides important clues for the mechanism of liver tumorigenesis.