Experimental Studies On Silencing PEG10 By RNA Interference For Strategy Of Hepatocellular Carcinoma

Objective Hepatocellular carcinoma (HCC) is one of the most common malignancies and cancer-related causes of death worldwide lacking the effective therapy. At present, surgical resection (segmentectomy, partial hepatectomy, hemihepatectomy or extend hemihepatectomy) or transplantation offers the only chance of cure of hepatocellular carcinoma. Surgical resection can achieve up 36~50% 5 year survival rates. Unfortunately, however, in most patients the tumors are unresectable because of multicentricity or poor hepatic function. In China the figure is less than 25 percent. The recurrence of new tumors after apparently successful resection remains a problem. Transplantation is indicated in patients with poor liver function and without extro-hepatic metastasis. Non-surgical treatments such as transcatheter arterial chemo embolization, percutaneous ethanol injection, radiofrequency ablation, microwave coagulation therapy and so on are limited. Gene therapy is a promising approach because it offers the potential of correcting the underlying genetic defect. Recently, evidence has been accumulated that the paternally expressed gene 10 (PEG10) is a promising target for cancer therapy. Several reports have indicated that PEG10 is specifically expressed in HCC. Our prior work showed that PEG10 is more specifically expressed in HCC than AFP. In this study we detect the level of PEG10 mRNA and protein expression in many kinds of human cancer cell lines involve hepatocellular carcinoma cell lines, pancreatic carcinoma cell lines, colorectal carcinoma cell line, breast carcinoma cell line and cervical carcinoma cell line. Our goal was to inhibit the PEG10 gene expression in HCC cell lines by siRNA, and investigate their suppressing efficacy of the PEG10 expression and their consequent antitumor potential in vivo and vitro. This would be a new useful method in gene therapeutic approach for hepatocellular carcinoma.METHODS: RT-PCR was employed to measure the PEG10 mRNA expression in cell lines. Western blotting was exerted to determine the level of proteins. Four potential siRNA target sites were identified on the PEG10 and inserted into the downstream of H1 promoter to recombine psiRNA-PEG10 plasmid. The four recombine psiRNA-PEG10 plasmids were named as psiRNA1, psiRNA2, psiRNA3 and psiRNA4, which were transiently transfected into HCC cell lines via Lipofectamine 2000 respectively. RT-PCR and Real-time RT-PCR were employed to measure the PEG10 mRNA expression. RT-PCR also was used to detect the lever of mRNA including Cyclin (cyclin D), CKI (p21, p27, and p16), CDK (CDK4) and E2F1. Western blotting was exerted to determine the level of PEG10, Cyclin, CKI, E2F1 and CDK protein. The cell growth was assessed by MTT assay and the apoptosis of HCC cells was evaluated by flow cytometry and confocal microscopy. HepG2 cell line was implanted subcutaneously into nude mice. Fourteen days after implantation, psiRNA2 and psiRNA-hH1-neo were injected into nude mice respectively. The tumor volume was measured. The histopathological changes of the tumors were observed by HE staining and electron microscope.RESULTS: The expression of PEG10 mRNA and protein was high in human hepatocellular carcinoma cell lines, pancreatic carcinoma and colorectal carcinoma cell lines but negative in other human tumor cell lines. The expression of PEG10 mRNA was remarkably inhibited at 48h after transfection, and psiRNA2 resulted in the highest inhibition rate. The level of mRNA CDKI (p21and p16) increased,whereas those of Cyclin, E2F1 and CDK decreased. HCC cell growth was also inhibited significantly (P<0.05). Flow cytometry analysis exhibited cell cycle was arrested at G0/G1 and the cell apoptosis rate reached the highest after psiRNA2 transfection, with significantly higher than that of the control group (P<0.01). Confocal microscopy revealed apoptotic cells. Western blotting showed the level of protein PEG10 and CDKI increased whereas those of E2F1, Cyclin and CDK decreased. After psiRNA2 and psiRNA-hH1neo were injected into nude mice, growth of hepatocellular carcinoma was significantly inhibited in the psiRNA2 therapy group as compared with that in the control group(P<0.001). The growth inhibitory rate was remarkably higher than that of the two control groups. Electron microscope revealed many apoptotic cells.CONCLUSIONS: PEG10 is highly expressed in hepatocellular carcinoma but not expressed in other human cancer besides digestive system carcinoma such as breast carcinoma and cervical carcinoma. Four eukaryotic expression vector of siRNA targeting PEG10 gene were successfully constructed and could inhibit the expression of PEG10 mRNA and protein in HCC cell lines. The inhibition of PEG10 by siRNA induced growth inhibition, apoptosis and cell cycle arrest at G0/G1. PsiRNA2 also results in marked inhibition of hepatocellular carcinoma growth in nude mice Therefore siRNA targeting PEG10 may become a novel promising strategy therapeutic of HCC.