miR-146a/b在Hep3B细胞中的表达及上下游调控机制探讨

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p-ERK1、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。

MiR-146a/b Expression and Related up and Down-stream Regulatory Mechanisms in Human Hepatoma Cells

Objective: To investigate the expression of miR-146a/b and related upstream regulatory mechanisms and downstream target proteins in human heptoma cells. Methods: The proliferation activities of the normal human hepatocyte cell line LO2 and human heptoma cell lines HepG2 and Hep3B were assayed by MTT. Total RNA and protein were extracted from the cells and the miR-146a/b levels were detected by RT-qPCR and protein levels of p-ERK, ERK, p-AKT, IκB, IRAK1, and TRAF6 were detected by Western blot. The activities of PI3K, ERK, AKT, and NF-κB signaling molecules were inhibited in Hep3B cells, respectively, and the corresponding expression levels of miR-146a/b and protein levels of IRAK1 and TRAF6 were detected. Results: The order of proliferation activities were Hep3B > HepG2 > LO2. Compared to LO2 and HepG2 cells, the expression levels of miR-146a and miR-146b increased significantly in Hep3B cells (P<0.01). Compared to LO2 cells, the expression levels of miR-146a decreased significantly in HepG2 cells (P<0.001). In Hep3B cells, the protein levels of p-ERK1, ERK1, p-AKT, IκB increased 10.87, 24.68, 6.67 and 1.92 times of LO2 cells, respectively; and the protein levels of IRAK1 and TRAF6 decreased 0.23 and 0.003 times of LO2 cells, respectively. In HepG2 cells, the protein levels of IκB and IRAK1 increased 4.46 and 2.69 times of LO2 cells, respectively. In Hep3B cells, inhibition of PI3K and AKT activity significantly reduced the expression levels of miR-146a/b at 12 h and 24 h (P<0.05). Inhibition of ERK and NF-κB activity had no effect on the expression levels of miR-146a/b at 12 h and 24 h. Inhibition of PI3K, AKT, ERK, and NF-κB signaling molecular activities elevated the TRAF6 and decreased the IRAK1 protein levels. Conclusions: PI3K/AKT signaling pathway down-regulates the protein levels of TRAF6 by up-regulating the expression levels of miR-146a/b in Hep3B cells. This mechanism provides valuable clue for researches on hepatocellular carcinogensis.