Wnt/β-Catenin信号通路在肝癌发生中的分子机制研究

The Study of Wnt /β-catenin Signaling Pathway in the Molecular Mechanism of Liver Cancer

目的探讨Wnt/β-Catenin信号通路在肝癌细胞增殖、迁移方面的影响和其在肝癌发生发展中的可能作用。方法体外培养HepG2和L02细胞,采用免疫荧光和Western Blot检测Wnt/β-catenin信号通路关键蛋白β-catenin、GSK3β、cyclin D1和c-myc蛋白的表达水平。培养的HepG2细胞分别用浓度100ng/ml的Wnt3α和20 ng/ml的DKK1处理,采用CCK-8检测细胞增殖活力,流式细胞实验检测细胞周期,Western Blot检测β-catenin、GSK3β、cyclin D1和c-myc蛋白的表达水平,Trans-well检测HepG2的迁移情况。结果与正常肝细胞L02相比,HepG2细胞高表达β-catenin,cyclin D1和c-myc,低表达GSK3β。Wnt3α能够显著促进HepG2细胞的增殖活力,而DKK1能够显著抑制其增殖。与Wnt3α组相比,DKK1处理组细胞G0/G1期细胞比例增加,(68.6±0.5)%VS(47.5±1.5)%(P<0.01),而S期细胞比例减少(17.4±0.5)%VS(28.6±0.5)%,(P<0.01)。Western Blot检测结果说明,Wnt3α提高了β-catenin、cyclin D1和c-myc的表达水平,抑制了GSK3β的表达,而DKK1抑制了β-catenin、cyclin D1和c-myc的表达。迁移实验透膜细胞数对照组为(176.40±12.98)、Wnt3α组为(238.20±18.38)、DKK1组为(110.40±9.46),P<0.05。结论 Wnt/β-catenin信号通路在促进HepG2细胞的增殖和迁移方面起重要作用,在肝癌的发生发展和转移中起着重要的作用,是肝癌发生的重要分子机制。

Objective To explore the effects of Wnt signaling pathway on proliferation and migration in HepG2 cells and its possible molecular mechanism in the occurrence and development of liver cancer. Meth- ods HepG2 and 1.02 cells were cultured in vitro. Immunofluorescence and western blot were used to detect the expression of the essential components of Wnt signaling β-catenin, GSK3β, eyelin D1 and c-myc. The cultured HepG2 cells were treated with 100ng/ml Wnt3a and 20ng/ml DKK1 in vitro. After treated with liT, morpho- logical of GES-1 ceils was observed using microscope. Then cell counting Kit-8 （ CCK-8 ） was used to detect the proliferation activity of HepG2 cells and flow eytometry assay was used .to assess cell cycle of HepG2 ceils. The expression of β-catenin, GSK3β, eyelin D1 and c-myc were detected using western blot aalysis. Trans-well as- say was used to assess the migration of HepG2 cells. Resdts Compared with L02 ceils, the expressions of β-catenin, cyclin D1 and c-myc in HepG2 cells increased significantly. But the expressions of GSK3β de- creased significantly. Compared with control group, the proliferation activity of HepG2 cells increased signifi- cantly after treated with Wnt3a, which was inhibited by DKK1 significantly. Compared with control group, HepG2 cells treated with DKK1 showed an increased proportion of cells at the GO/G1 phase, （68.6± 0.5）% VS （47.5 ±1.5）%, （P 〈0.01） and decreased proportion of cells at the S phase, （17.4 ±0.5）% VS （28.6 ±0.5）% （P 〈0.01 ）. Western blot results demonstrated that Wnt3a increased the expression of ^-catenin, cyc- lin D1 and c-myc in HepG2 cells. However, DKK1 inhibited these proteins expression. The trans-well assay results indicated that the penetrated ceils in control group was 176.40 ± 12.98, Wnt3a group was 238.20 ± 18.38, and DKKI group was 110.40 ± 9.46 （ P 〈 0.05 ）. Conclusions Wnt/β-catenin signaling pathway promotes the proliferation and migration of HepG2 ceils. Wnt/β-catenin signaling pathway may be an origin mo- lecular mechanism of liver cancer which plays an essential role in the occurrence and development of liver cancer.