5-ethynyl-2--deoxyuridine and Liver-Neoplasms

To explore the silencing glypican-3 (GPC-3) gene transcription by specific small hairpin RNA (shRNA) on the inhibition of hepatoma cells with high metastatic potentiality and hepatoma growth.. After MHCC-97H cells were transfected with higher effective GPC-3-shRNA, GPC-3 mRNA was analyzed by multiple FG-RT-PCR or protein by Western blot. Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine and sulforhodamine B assay, its migratory metastasis and invasiveness by wound healing or transwell chamber system and cell apoptosis was detected by Caspase-Glo(®) 3/7 Luminescence assay. Nude mice were subcutaneously injected with stable MHCC-97H cells for observing the forming time or volume of xenograft tumors. And the expressions of GPC-3, β-catenin, p-GSK3β and CyclinD1 were analyzed by immunohistochemistry.. After shRNA1 transfection with high efficiency (>80%), the expression of GPC-3 was down-regulated to 75.6% (t = 15.473, P < 0.001) at mRNA level in accordance with its protein, inhibiting cell proliferation (71.1%, t = 10.468, P < 0.001) notably, decreasing its migration (80.1%, t = 32.697, P < 0.001) and invasiveness (69.1%, t = 39.647, P < 0.001). β-catenin was down-regulated (67.7%, t = 18.4, P < 0.001) and Gli1 increased (53.5%, t = -4.824, P = 0.008) with its protein. The average forming time of subcutaneous tumors was 11.2 days (d) in the shRNA group and it was significantly longer (P < 0.01) than that in the control (5.3 d) or shRNA-neg (5.5 d) group. And the average volume (65.5 mm(3)) of tumors with decreased GPC-3, β-catenin, p-GSK3β, and cyclinD1 expressions in the shRNA group was significantly smaller (P < 0.01) than that in the shRNA-neg (365.7 mm(3)) or control (404.8 mm(3)) group, respectively.. Specific shRNA might intervene effectively the GPC-3 gene transcription and inhibit invasion and tumor growth. Thus GPC-3 may become a potential molecular target for hepatoma gene therapy.

Topics: Animals; Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Proliferation; Deoxyuridine; Down-Regulation; Gene Silencing; Glypicans; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Messenger; RNA, Small Interfering; Transcription, Genetic; Transfection