下调GPC-3转录经Wnt/β-catenin通路对肝细胞癌生长的抑制作用

Down-regulating GPC-3 transcription via Wnt/β-catenin pathway for inhibiting hepatocellular carcinoma growth

目的研究干预磷脂酰肌醇蛋白多糖-3(glypican-3, GPC-3)活化经Wnt/β-catenin通路对肝细胞癌(hepatocellular carcinoma, HCC)的抑制作用。方法将miRNA干扰质粒转染GPC-3高表达的HepG2和Hep3B细胞,以CCK-8法检测细胞增殖,平板法分析克隆形成,以流式细胞术分析细胞周期;以裸鼠移植瘤模型观察成瘤时间、体积、生长曲线;以免疫组化法和免疫印迹法分析GPC-3及Wnt/β-catenin通路关键分子β-catenin、p-GSK3β及Cyclin D1的变化。结果特异性miRNA成功转染HepG2和Hep3B细胞后,体外研究显示HCC细胞增殖受到抑制、克隆形成明显降低和发生G1期阻滞,差异有显著统计学意义(P<0.01),β-catenin及p-GSK3β显著降低(P<0.01);体内研究显示,移植瘤成瘤时间延长(t=12.89,P<0.01)、瘤体小(t=15.29,P<0.01);瘤内GPC-3表达明显下降(t=6.67,P<0.01)、相关信号分子β-catenin(t=9.61,P<0.01)、p-GSK3β(t=4.81,P<0.01)及Cyclin D1(t=5.90,P<0.01)表达均显著下调。结论干预GPC-3活化经Wnt/β-catenin通路抑制HCC细胞增殖和生长,提示GPC-3为HCC治疗的潜在靶目标。

Objective To down-regulate glypican-3(GPC-3) via Wnt/β-catenin pathway on effects of hepatocellular carcinoma(HCC) growth. Methods Specific miRNA plasmids were transfected into HepG2 and Hep3B cell lines with strongest GPC-3 expression and then cell proliferation was tested by CCK-8 kit. Clone formation was checked by plate clone formation. Alteration of cell cycle was measured by flow cytometry. Nude mice xenograft models were made, tumor growth was daily monitored, GPC-3, cell cycle regulator Cyclin D1, and key molecules in Wnt/β-catenin pathway were analyzed by Western blotting or immunohistochemistry. Results Specific miRNA was transfected into HepG2 or Hep3B cells with GPC-3 overexpression in vitro. GPC-3 was markedly down-regulated with inhibiting cell proliferation in a time-dependent manner, decreased not only in clonogenic capacity in plate(P<0.01) with cell cycle arrested at G1 phase(P<0.01), but also significantly decreasing(P<0.01) of β-catenin or p-GSK3β of Wnt/β-catenin pathway. Silencing GPC-3 in HepG2 cells affected the growth of nude mice xenografts in vivo(t=12.89, P<0.01), smaller tumors(t=15.29, P<0.01) with less GPC-3(t=6.67, P<0.01),β-catenin(t=9.61, P<0.01), p-GSK3β(t=4.81, P<0.01), and Cyclin D1(t=5.90, P<0.01). Conclusion Intervening GPC-3 inhibits HCC growth via Wnt/β-catenin pathway in vitro or in vivo, and indicates that GPC-3 should be a novel targeted-molecule for HCC.