Yu Dan-dan, Yao Min, Chen Jie, Wang Li, Yan Mei-juan, Gu Xing, Qiu Li-wei, Dong Zhi-zhen, Yao Deng-fu, Lu Shao-lin
Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
Zhonghua Gan Zang Bing Za Zhi. 2013 Jun;21(6):452-8. doi: 10.3760/cma.j.issn.1007-3418.2013.06.016.
To construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms.
GPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2, MHCC-97H, and Huh7. shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting, respectively. The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays, on migration by wound healing (scratch) assay, on invasion by transwell chamber assay, and on apoptosis by luminescence assay of caspase-3/7 activity. The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor, GLI1, and the beta-catenin Wnt signaling factor, b-catenin), immunofluorescent staining (for the insulin-like growth factor-II, IGF-II), and ELISA (for the vascular endothelial growth factor, VEGF). Pairwise comparisons were made by the independent sample t-test, and multiple comparisons were made by one-way ANOVA.
In all cell lines, transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (% reduction as compared to the non-target control shRNA: HepG2, 89.2+/-6.0%, t = -25.753, P less than 0.001; MHCC-97H, 75.3+/-4.9%, t = -26.487, P less than 0.001; Huh7, 73.6+/-4.6%, t = -27.607, P less than 0.001); the GPC-3 protein levels were similarly reduced. The GPC-3 shRNA-silenced cells showed significantly reduced proliferative, migratory and invasive capacities, as well as significantly increased apoptosis. The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of b-catenin mRNA (HepG2, 46.9+/-0.6%; MHCC-97H, 67.5+/-2.7%; Huh7, 56.3+/-8.4%) and significant up-regulation of GLI1 mRNA (HepG2, 49.2+/-28.6%; MHCC-97H, 54.6+/-24.4%; Huh7, 31.6+/-15.7%). At 72 h after transfection, the HepG2 cells showed significant down-regulation of VEGF protein (54.3+/-1.5%, t = 46.746, P less than 0.001).
GPC-3 contributes to migration, invasion, angiogenesis, and apoptosis of hepatoma cells, possibly through its interactions with the Wnt/b-catenin and Hedgehog signaling pathways. GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.
构建磷脂酰肌醇蛋白聚糖-3（GPC-3）短发夹RNA（shRNA），并研究GPC-3转录沉默对肝癌细胞侵袭和血管生成机制的影响。
构建GPC-3特异性shRNA和非靶向对照shRNA，并将其转染到人肝癌细胞系HepG2、MHCC-97H和Huh7中。分别通过荧光定量逆转录（FQRT）-PCR和蛋白质印迹法在mRNA和蛋白质水平上确认shRNA介导的GPC-3表达沉默。通过EdU和磺基罗丹明B测定法检测沉默的GPC-3表达对细胞增殖的影响，通过伤口愈合（划痕）测定法检测对迁移的影响，通过Transwell小室测定法检测对侵袭的影响，通过caspase-3/7活性发光测定法检测对凋亡的影响。通过FQRT-PCR（用于胶质瘤相关癌基因同源物-1刺猬信号因子GLI1和β-连环蛋白Wnt信号因子β-连环蛋白）、免疫荧光染色（用于胰岛素样生长因子-II，IGF-II）和ELISA（用于血管内皮生长因子，VEGF）检测沉默的GPC-3表达对血管生成相关信号因子的影响。采用独立样本t检验进行两两比较，采用单因素方差分析进行多组比较。
在所有细胞系中，用GPC-3特异性shRNA转染均显著降低了GPC-3 mRNA水平（与非靶向对照shRNA相比降低的百分比：HepG2，89.2±6.0%，t = -25.753，P＜0.001；MHCC-97H，75.3±4.9%，t = -26.487，P＜0.001；Huh7，73.6±4.6%，t = -27.607，P＜0.001）；GPC-3蛋白质水平也同样降低。GPC-3 shRNA沉默的细胞显示出增殖、迁移和侵袭能力显著降低，以及凋亡显著增加。shRNA介导的GPC-3沉默伴随着β-连环蛋白mRNA的显著下调（HepG2，46.9±0.6%；MHCC-97H，67.5±2.7%；Huh7，56.3±8.4%）和GLI1 mRNA的显著上调（HepG2，49.2±28.6%；MHCC-97H，54.6±24.4%；Huh7，31.6±15.7%）。转染后72小时，HepG2细胞显示VEGF蛋白显著下调（54.3±1.5%，t = 46.746，P＜0.001）。
GPC-3可能通过与Wnt/β-连环蛋白和刺猬信号通路相互作用，促进肝癌细胞的迁移、侵袭、血管生成和凋亡。GPC-3可能是基于分子的治疗方法进行基因沉默治疗肝细胞癌的一个有用靶点。
