miR-198 通过靶向ZEB2 调控EMT 过程抑制肝癌细胞增殖和迁移的机制研究

目的 探讨微小核糖核酸-198（miR-198）在肝细胞癌（hepatocellular carcinoma，HCC）组织细胞中的表达及其对癌细胞增殖、迁移的影响及其相关机制。方法 采用qRT-PCR 检测miR-198 在HCC 组织及细胞(HepG2，Hep3G，MHCC97H，Huh7) 中的表达情况；采用脂质体2000 向Huh7 细胞中单独转染miR-198 mimics，共转染miR-198 mimics 和pcDNA3.1-ZEB2；生物信息学网站预测miR-198 的潜在靶基因，采用双荧光素酶报告基因实验进行验证；采用Western blot 检测E 盒结合锌指蛋白2（E-box binding zinc finger protein 2, ZEB2）及上皮-间质转化（epithelialmesenchymaltransformation, EMT）相关蛋白表达；CCK-8 法和细胞划痕实验检测Huh7 细胞增殖和迁移能力。结果 20 例HCC 组织中miR-198相对表达量（0.354±0.022）明显低于邻近正常组织（4.762±1.135），差异有统计学意义（t=17.365，P＜ 0.001）。HCC细胞系HepG2（0.589±0.103），Hep3G（0.495±0.086），MHCC97H（0.558±0.056）和Huh7（0.362±0.045）中miR-198 相对表达量较正常肝细胞LO2（1.823±0.125）显著降低（t=17.159，18.466，17.590，20.315，均P ＜ 0.05）。miR-198 mimics 组细胞增殖（0.398±0.146 vs 0.691±0.213）和迁移能力（20.012±2.103 vs 84.032±6.512）较对照组明显降低（t=1.965，52.459，均P ＜ 0.001）。miR-198 mimics 组细胞中E-cadherin 表达较对照组明显升高，N-cadherin和Vimentin 表达较对照组明显降低（t=18.478，17.550，19.706，均P ＜ 0.01）。ZEB2 是miR-198 的靶基因，miR-198负调控ZEB2。20 例HCC 组织中ZEB2 相对表达量（3.621±1.143）明显高于邻近正常组织（ 0.736±0.030），差异有统计学意义（t=11.284，P ＜ 0.001），与miR-198 表达呈负相关（r=-0.702，P ＜ 0.05）。共转染过表达ZEB2 逆转了miR-198 mimics 对HCC 细胞增殖、迁移和EMT 的抑制作用。结论 miR-198 在HCC 组织和细胞中表达下调，miR-198 通过靶向负调控ZEB2 的表达抑制Huh7 细胞的增殖和迁移。

Study on the Mechanism of miR-198 Inhibiting the Proliferation and Migration of Hepatoma Cells by Regulating EMT Process by Targeting ZEB2

Objective To investigate the expression of micro RNA（miR）-198 in hepatocellular carcinoma (HCC) tissues and its effects on the proliferation and migration of cancer cells and related mechanisms. Methods qRT-PCR was used to detect the expression of miR-198 in HCC tissues and cells (HepG2, Hep3G, MHCC97H, Huh7). The liposome 2000 was used to transfect miR-198 mimics alone，co-transfect miR-198 mimics and pcDNA3.1-ZEB2 into Huh7 cells. Bioinformatics website predicted that the potential target gene of miR-198，and the double luciferase reporter gene experiment was used for verification. Western blot was used to detect the expression of E-box binding zinc finger protein 2（ZEB2） protein and epithelial-mesenchymal transformation (EMT) related proteins,and CCK-8 and scratch test were used to detect the proliferation and migration ability of Huh7 cells. Results The relative expression of miR-198 in 20 HCC tissues (0.354±0.022）was significantly lower than that in adjacent normal tissues（4.762±1.135), with the difference was statistically significant (t=17.365, P ＜ 0.001). HCC cell lines HepG2（0.589±0.103）, Hep3G（0.495±0.086）, MHCC97H（0.558±0.056） and Huh7（0.362±0.045） was significantly lower than that in normal liver cells LO2 miR-198 in normal liver cell（1.823±0.125）（t=17.159, 18.466, 17.590, 20.315, all P ＜ 0.05）. The proliferation(0.398±0.146 vs 0.691±0.213) and migration(20.012±2.103 vs 84.032±6.512) of miR-198 mimics group were significantly decreased compared with the control group (t=1.965, 52.459, all P ＜ 0.001). The expression of E-cadherin(2.010±0.089 vs 1.001±0.032) in miR-198 mimics group was significantly higher than that in control group, and the expression of N-cadherin(0.362±0.056 vs 1.001±0.029) and Vimentin(0.356±0.048 vs 1.000±0.030) was significantly lower than that in control group (t=18.478, 17.550, 19.706, all P ＜ 0.01). ZEB2 was the target gene of miR-198 and miR-198 negatively regulates ZEB2. The relative expression of ZEB2 in 20 HCC tissues (3.621±1.143)was significantly higher than that in adjacent normal tissues（0.736±0.030) ，the difference was statistically significant (t=11.284, P ＜ 0.001), and was negatively correlated with miR-198 expression (r= -0.702, P ＜ 0.05). Overexpression of ZEB2 by cotransfection reversed the inhibitory effect of miR-198 mimics on HCC cell proliferation, migration and EMT. Conclusion MiR-198 was down-regulated in HCC tissues and cells, and inhibits the proliferation and migration of Huh7 cells by negatively regulating the expression of ZEB2.