| Background and aim:Liver cancer is one of common malignant tumors in China, with the extremely high mortality rate. The mortality rate high priority in cancer mortality, third only to the stomach, esophagus cancer; in most parts of the rural areas accounted for the second place, second only to gastric cancer.110,000 people died of liver cancer every year in our own country, which is up to 45% of total number of people who died of liver cancer all over the world.The pathogenesis of liver cancer is contributed to a few of factors including viral hepatitis,flavacin and hereditary alteration etc, which may lead to the oncogene overexpression and the lost expression of tumor suppressor and finally caused the genesis of liver cancer. Hepatoma-derived growth factor (HDGF), a novel growth factor, has a widely expression in many normal cells and tumor cells. It plays an important role in cell proliferation, differentiation and angiogenesis. It is considered as a promising marker for predicting the invasion, matastasis and prognosis of carcinomas including liver cancer in clinical researches. In this study, we firstly tested the mRNA expression in liver cancers and their correponding noncancerous tissues, as well as HepG2 cells. Subsequently, we investigate the HDGF expression in hepactocellular carcinoma (HCC) and its correlation with clinicopathologic features, including the survival of patients with HCC. Further, we examined the biological processes regulated by HDGF during the development of hepactocellular carcinoma (HCC) using HepG2 cell line as a model system.Method:We collected 10 paired liver cancers and their corresponding noncancerous tissues and then extracted their total RNA by route Trizol method.In addition,we also extracted total RNA of HepG2 cells in the same way. Subsequently, these total RNAs were transcripted into cDNA, which were used to examine the mRNA expression of HDGF in liver cancers and their corresponding noncancerous tissues and HepG2 cells by real time qPCR. we used immunohistochemistry to compare HDGF protein expression in Hepatocellular carcinoma (HCC)(137cases) and normal liver tissues (49 cases) and further analyze the HDGF protein expression in clinicopathologically characterized 137 HCC cases. In the next step, we designed the shRNA sequences specifically targeting to HDGF by online program of Invitrogen Inc and recombinated them into pLenti6/BLOCK-iTTM-DEST lentivirus expression vector.Sequencing analysis was used to identify the correction of shRNA.Further,We transfected these pLenti6/BLOCK-iTTM-DEST lentivirus expression vector into HepG2 cells. After screening by blasticidin, positive single clone cells were displayed in culture plate in the 14th day. After these cells were further expanded to culture, total RNAs of these cells were extracted and then transcripted into cDNA. real time qPCR was used to examine the interference efficiency of HDGF mRNA expression. Western blot was utilized to finally comfirm the HDGF protein level after HDGF was silenced by siRNA method. Following the successful establishment of stable cells, we examined in vitro cell growth by MTT assay, anchorage-independent growth by soft-agar colony formation assay, cell migration,invasion by transwell and boyden chamber assays, and in addition, the in vivo tumor growth by xenograft transplantation of HepG2 cells into nude mice.Result:We used route Trizol method to successfully extract total RNA of liver cancers and their corresponding noncancerous tissues and HepG2 cells. The expression level of HDGF mRNA is highly expressed in all liver cancer tissues and HepG2 cell lines cpmpared with the noncancerous tissues by real time qPCR assay. Immunohistochemistry results showed that protein expression level of HDGF was markedly higher in HCC tissues than that in the normal liver tissues(P=0.011). In addition, high expression of HDGF protein was positively correlated with T classification((p=0.000),N classification (p=0.000) and clinical stage (p=0.000) of HCC patients. Patients with higher HDGF expression showed a significantly shorter overall survival time than did patients with low HDGF expression. Multivariate analysis suggested that HDGF expression might be an independent prognostic indicator(0.000) for the survival of patients with HCC. Subsequently, we successfully constructed the pLenti6/BLOCK-iTTM-DEST lentivirus shRNA expression vector specifically targeting to HDGF. The result of sequence analysis showed the consistency with the designed shRNA sequence.After pLenti6/BLOCK-iTTM-DEST lentivirus shRNA-HDGF expression vectors were transfected into HepG2 cells, we established the HepG2 cells with stably knocking down the endogenous expression level of HDGF. After examined by real time qPCR, the lowest expression level of HDGF was shown in HepG2-shRNA3-HDGF cells and the inhibition efficiency was up to 80% compared with HepG2-shRNA-Ctr cells.The western blot result also displayed that the level of HDGF protein expression in HepG2-shRNA3-HDGF cells was only 24.7% compared with HepG2-shRNA-Ctr cells.which demonstated that HDGF-specific shRNA (shHDGF) successfully knocked down its endogenous expression in HepG2 cells. Compared to the parental HepG2 and control shRNA-transfected (shCtrl) HepG2 cells, knocking down the expression of HDGF not only exhibited significantly the reduced ability of in vitro cell growth, anchorage-independent growth, cell migration and invasion (P<0.05), but also indicated markedly the decreased ability of in vivo the xenograft transplants from shRNA-HDGF cells giving rise to much smaller tumors as compared to those from shRNA-Ctrl cells.Conclusion:HDGF is highly expressed in liver tissues and HepG2 cell lines.Overexpressed HDGF is an independent prognostic factor in HCC patients. High HDGF expression is associated with poor overall survival in patients with HCC. We successfully construct the pLenti6/BLOCK-iTTM-DEST lentivirus shRNA expression vector that can specifically suppress the expression of HDGF in HepG2 cells. Knocked down the expression of HDGF markedly inhibits the growth, anchorage-independent growth, migration and invasion of HepG2 cells. HDGF may be an favourable target for gene therapy in liver cancer. |
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