| Objective The genesis and development of hepatocellular carcinoma(HCC) is a multi-stage complex processes involving the chromosome aberrations, the gene mutations, the epigenetic alterations, and abnormal regulation of multiple signaling pathways. Recently, oncofetal glypican-3(GPC-3) that links to cell membrane surface with a glycosyl-phosphatidy-linositol anchor is a specific molecular marker of hepatocyte malignant transformation, and now, it has been applied in HCC diagnosis. GPC-3 promotes HCC growth and metastasis through stimulation of multiple signaling pathways such as Wnt, and the suppression of its activation could significantly inhibit hepatoma cell proliferation. GPC-3 gene might become a potential effective target for HCC therapy and its molecular mechanism need to be clarified. In the present study, the two models of hepatoma cells in vitro and nude mice subcutaneously xenograft tumors in vivo were used to investigate the down-regulation of GPC-3 gene at m RNA level by specific mi RNA on effects of hepatoma cell proliferation or hepatoma growth, and analyze the key molecule expressions of the Wnt/β-catenin pathway for exploring its molecular mechanism.Methods Expression of GPC-3 in human hepatoma SMMC-7721, MHCC-97 H, Bel-7402, PLC/PRF/5, Hep G2, Hep3 B, and Bel-7404 cell lines were analyzed by western blotting, and the Hep G2 and Hep3 B cell lines with strongest GPC-3 were selected for further analysis. Highest efficiency p CMV-GPC-3-mi RNA plasmids were successfully constructed and screened, confirmed by sequencing, and then transfected Hep G2 and Hep3 B cell lines, interference efficient was quantitatively evaluated by a fluorescence quantitation polymerase chain reaction assay. Expression of GPC-3, cell cycle regulator Cyclin D1, and key molecules in Wnt/ β-catenin pathway were analyzed by Western blotting or immunohistochemistry. Cell proliferation was tested by CCK-8 kit. Clone formation rate is checked by plate clone formation or soft agar clone formation assay. Alteration of cell cycle was measured by flow cytometry. Stable cell clones were screened by blasticidin and used for nude mice xenograft tumor models. Tumor growth was daily monitored, and histopathology was examined with Hematoxylin and Eosin(H & E) staining.Results Different GPC-3 expressions in human HCC Hep G2, SMMC-7721, MHCC-97 H, Bel-7402, PLC/PRF/5, Hep3 B, and Bel-7404 cell lines were confirmed by Western blotting, and the Hep G2 or Hep3 B cells were the strongest GPC-3 expression among them. Four kinds of interference p CMV-GPC-3-mi RNA plasmids were constructed, screened, confirmed by sequencing, and the most efficiency or specific plasmid(mi RNA-1) was transfected into the Hep G2 or Hep3 B cells with GPC-3 overexpression in vitro, respectively. The GPC-3 expression in both cells was markedly down-regulated(P < 0.01) at m RNA or protein level with inhibiting cell proliferation in a time-dependent manner, and more inhibiting efficiency(P < 0.01) was found in the serum-free cultures compared with those in the neg-mi RNA or the untreated group. Both cells with mi RNA1 stable transfection significantly decreased not only in clonogenic capacity in plate(P < 0.01) but also in soft agar(P < 0.01) with cell cycle arrested at G1 phase and the greatly decreasing Cyclin D1 expression(P < 0.01). The key molecules of Wnt/β-catenin pathway were significantly down-regulated in the β-catenin(t = 23.24, P < 0.01 or t = 14.43, P < 0.01) or the p-GSK3β(t = 13.88, P < 0.01 or t = 35.52, P < 0.01) in the Hep G2 or Hep3 B cells compared with those in the neg-mi RNA or untreated group. Silencing GPC-3 gene transcription in the Hep G2 cells effected the growth of nude mice xenograft tumors in vivo(11.17 ± 0.98 d vs 5.33 ± 0.52 d, t = 12.89, P < 0.01) and the tumor volume was significantly smaller(65.48 ± 13.66 mm3 vs 404.83 ± 52.63 mm3, t = 15.29, P < 0.01) than those in the untreated group. Tumor cells in the mi RNA group were funicular or platy with comparatively clear boundaries. Dark staining pyknotic nuclei was found occasionally and the tumor tissues had relatively complete plasmalemma examined by H & E staining. The expressions of tumor 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) in the mi RNA group were significantly decreased than those in the untreated group, and no statistics difference between the neg-mi RNA and the untreated group were confirmed by immunohistochemical analysis.Conclusions Intervencing GPC-3 gene transcription inhibited HCC cell proliferation and heptoma growth through Wnt/β-catenin signaling pathway in vitro or in vivo, and suggesting that GPC-3 should be a novel therapeutic target for HCC. |
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