Targeted Glypican-3Gene Transcripation By Short Hairpin RNA In Inhibition Of Hepatoma Cell Proliferation In Vitro

Objective Hepatocellular carcinoma (HCC) is characterized by multi-cause, obvious multi-stage and multi-gene complex process, which includes multiple genetic alterations such as activation of oncogenes, inactivation of tumor suppressor genes, and activation of complex signaling pathways. Its prognosis is very poor, and early diagnosis and efficacious therapy are of the utmost importance. Recently, Glypican-3(GPC-3) with carcinoembryonic antigen has been confirmed that its expression is closely associated with hepatocyte malignant transformation and a promising biomarker for HCC diagnosis with high specificity and sensitivity. However, whether GPC-3becoming an effective molecular-targeted for HCC gene therapy or not remains to be explored. The objectives of the present study were to investigate the silencing GPC-3gene transcription on the effects of hepatoma cell proliferation, invasion and apoptosis by constructing and screening GPC-3short hairpin RNA (shRNA). Further to explore the mechanism of GPC-3promotes hepatoma cell proliferation by Wnt and Hh signaling, and to evaluate the effect of silencing GPC-3on the sensitivity of multitarget chemotherapy.Methods Four pairs of shRNA targeting different sites of GPC-3gene were constructed according to its sequence, and confirmed by sequencing. The shRNAl-4plasmids were transfected into HepG2, MHCC-97H, and Huh7cell ines.The highest efficiency plasmid was identified by Fluorescence quantitative everse transcriptase-polymerase chain reaction at gene transcription and Western blotting at protein level, respectively, which is also examinate the change of expression about β-catenin and Gli1. The proliferation of HepG2cells was texted by MTT assay. EdU and Hoechst33342staining were used to evaluate the proliferation and apoptosis of hepatoma cell. Cell survival, migration and invation were investigated by Sulforhodamine B assay, Wound-healing, and Transwell chamber assay, respectively. Enzyme-linked immuno-adsordent assay was used to analyze the quantitative expression of vascular endothelial growth factor (VEGF) of HepG2cells. Alteration of cell cycle and HepG2cell apoptosis were detected by flow cytometry, Annexin-V-PE/7-AAD double staining assay and DNA-ladder electrophoresis, respectively. The activity of Caspase3/7was analyzed for exploring the mechnism of hepatoma cell apoptosis.Results GPC-3-shRNAl-4plasmids were successfully constructed, and confirmed by sequencing. The rate of HepG2, MHCC-97H, Huh7cells transfected with shRNA were all up to80%. The rate of silencing GPC-3mRNA by shRNAl was89.3%(t=-25.753, P<0.001).75.6%(t=15.473, P<0.001) and73.8%(t=15.004, P<0.001), respectively, which accordance with GPC-3protein down-regulation tested by Western blotting. Photographs from EdU assay can clearly indicated that the proliferation of the hepatoma cell was inhibited notably by shRNAl. The proliferation rate of the transfected HepG2cells was28.9%in the GPC-3shRNAl group, and20.5%in the GPC-3shRNAl plus with sorafenib (100μM) group, respectively. Meanwhile, it has time-dependent manner. The cell cycles after the cells transfected with shRNAl were arrested in the G1phase, and the apoptosis rate of HepG2cells was up to66.8%(t-11.456,P=0.008). The inhibition of proliferation, migration, and invation were separately up to80%and50%in hepatoma cell by shRNAl. Silencing GPC-3by shRNAl notably down-regulation β-catenin (46.9%,67.7%,55.9%) and up-regulation Gli1(47.4%,53.3%,31.4%) in mRNA and protein level in HepG2, MHCC-97H, and Huh7cells, respectively. Photographs from immunofluorescent staining demonstrated that β-catenin and IGF-Ⅱ were significantly decreased in transfected cells compared to the control group. The most efficiency IC50values of sorafenib, rapamycin, and erlotinib were4.67±1.20μM,7.85±2.00nM, and18.38±0.56μM in HepG2cells of hepatoma cell. The rate of proliferation inhibition in cells, which were co-treated with shRNAl and sorafenib, rapamycin, erlotinib, were higher than transfected alonely. The VEGF expression was decreased up to36.8%,46.2%and54.23%in HepG2cells at24h,48h and72h, respectively. When HepG2cells were combinationed with shRNAl and sorafenib, rapamycin, erlotinib, the apoptosis rate of HepG2cells was up to three times than control group, but when pretreatmented with Z-VAD-FMK, the rate were decreased to31.5%sharply.Conclusions Specific shRNA might intervene effectively GPC-3gene transcription, inhibit hepatoma cell proliferation, migration, invasion, and inducing apoptosis with β-catenin positive or Glil negative regulator, down-regulate IGF-Ⅱ and VEGF expression, have synergetic effect with multitarget chemotherapy, and promote apoptosis through Caspase pathway, suggesting that GPC-3should be a potential target gene for hepatoma gene therapy.