Intervention Of IGF-i Receptor Activation By ShRNA In Inhibition Of Hepatoma Cell Proliferation And Its Molecular Mechanism

Objective The pathogenesis of hepatocellular carcinoma(HCC) includes viruses, chemical carcinogenesis, and other multiple risk factors resulting in oncogene activation, tumor suppressor gene inactivation, and some genes reactivation in embryonic stages from starting, promoting to evolving in hepatocarcinogenesis. Recently, the overexpression of insulin-like growth factor-I receptor(IGF-IR) is closely associated with hepatocyte malignant transformation. However, the molecular mechanism of its abnormal expression and whether becoming a new valuable target for HCC gene therapy remains to be explored. 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 IGF-IR gene at m RNA level by specific sh RNA on effects of the cell proliferation, cell cycle, apoptosis, and the synergistic role with anti-cancer drugs; and to confirm the inhibiting efficiency of the xenograft tumor formation or growth through interferencing IGF-IR gene transcription.Methods Human hepatocyte(LO2), hepatoma PLC/PRF/5, Hep G2, and Bel-7404 cell lines were used for the present study. Theconstructed plasmids contained 4 pairs of sh RNAs targeting different sites of IGF-IR gene and 1 pair of negative sh RNA were inserted into p GPU6/GFP/Neo vector, transfected into the hepatoma cells, and screened the most effective one confirmed at m RNA level by real time fluorescent quantitation PCR. Expressions of IGF-IR and cyclin D1 were confirmed by Western blotting. Cell proliferation or survival combining with and without chemotherapy or targeted drug was examined by cell counting kit-8. The cell cycle or apoptosis was analyzed by flow cytometry or Annexin-V-PE/ 7-ADD in vitro. Nude mice were divided into control, neg-sh RNA, and sh RNA group, injected s. c. in the shoulder-backs with 0.2 ml of the transfected hepatoma cell(2×107/mouse) suspension for the xenograft tumor growth, sacrificed at end of 5 weeks, harvested, and analyzed. Morphological alteration of tumor was confirmed by H&E staining, tumor IGF-IR expression was analyzed by immunohistochemistry.Results The different expression of cellular IGF-IR was presented among LO2 and hepatoma cells. The level of IGF-IR expression in PLC/PRF/5 or Bel-7404 cells was higher than that in LO2 or Hep G2 cells. After screening, the IGF-IR-sh RNA4 was the most efficiency for interferencing IGF-IR gene transcription with specificity among 4 pairs of the successful constructed plasmids. The sh RNA4 transfection efficiency was 71% in PLC/PRF/5 cells and 90% in Bel-7404 cells with inhibited 59.6 ± 2.8% and 54.9 ± 2.6% at m RNA level those in accordance with the down-regulation of IGF-IR protein, respectively. After the cells transfected with sh RNA4 at 72 h, the inhibition rate of cell proliferation was 61.47 ± 1.7% in Bel-7404 cells(t = 5.493, P < 0.005) and 63.87 ± 3.9%(t = 19.244, P < 0.001) in PLC/PRF/5 cells in time dependent manner. Meanwhile, the cell cycles were arrested in the G1 phase, and the expression of cellular cyclin D1 was significantly down-regulatedwith increasing cell apoptosis. Besides, when the combining application of specific sh RNA4 plus sorafenib or oxaliplation, the synergistic inhibition effects were found on the cell survival or cell proliferation with the drug IC50 values decreased markedly. The xenograft tumor models were successfully made with the nude mice injected with the transfected cells. The tumor-forming time(14.0 ± 1.1 days) in the sh RNA group was significantly lengthened than that in the control(7.2 ± 0.75 days,t = 12.593, P < 0.001) or the neg-sh RNA(7.5 ± 1.0 days,t = 10.710, P < 0.001) group; The tumor volume(143 ± 24 mm3) in the sh RNA group was significantly smaller than that in the control(372 ± 46 mm3, t = 10.776,P < 0.001) or the neg-sh RNA(350 ± 50 mm3, t = 9.142, P < 0.001) group. Compared with the sh RNA group, the tumor morphological alteration in the control group was audio-videoailable atypia confirmed by H&E staining and the level of IGF-IR expression showed strongly positive in the control or neg-sh RNA group and only weakly positive in the sh RNA group.Conclusions Silencing IGF-IR gene transcription inhibited significantly hepatoma cell proliferation, induced apoptosis and enhanced the cell sensitivity to targeted or chemotherapy drug in vitro, and xenograft tumor growth in vivo, suggesting that IGF-IR might be a new potential target for hepatoma gene therapy.