Recombinant Adenovirus Induced RNAi Suppress Expression Of Epidermal Growth Factor Receptor (erbB1) In HepG2 Cells

Background and Objective:Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world and is estimated to cause half a million deaths annually. The incidence of HCC is dramatically increasing in the USA, Europe and Asia, due to high prevalence of chronic hepatitis B and hepatitis C virus infections, alcohol disease, diabetes and obesity. Unfortunately, the majority of patients suffer from advanced disease at presentation. Therefore curative local ablation, surgical resection of HCC, or liver transplantation can be achieved only in a minority of patients. Local tumor destruction, chemoembolization or systemic chemotherapy are the treatment options of advanced HCC. However, overall survival is poor. Apart from chemoembolization, which improves survival in well-selected patients with unresectable HCC, palliative treatment options do not appear to greatly improve overall survival. Therefore, innovative treatment approaches are urgently needed. Recently, evidence has been accumulated that the epidermal growth factor receptor (EGFR) is a promising target for cancer therapy. A great variety of tumors show abnormal, enhanced and/or constitutive expression of EGFR. Several reports indicate that EGFRs are expressed frequently in human HCC, most likely contributing to the aggressive growth characteristics of these tumors. Especially in poorly differentiated HCCs, EGFR overexpression has been demonstrated to be a negative prognostic factor, since it positively correlated with early tumor recurrence and the occurrence of extrahepatic metastasis. Hence, the EGFR is a promising target for innovative treatment strategies in HCC. Epidermal growth factor receptor (EGFR or erbB1) is a glycoprotein with a molecular weight of 170 000 with an intrinsic tyrosinespecific protein kinase, which is stimulated upon EGF binding. Activation of EGF receptor tyrosine kinase results in the generation of a number of intracellular signals that culminate in cell proliferation. The known downstream effectors of EGF receptor include PI3-K, RAS-RAF-MAPK signal pathways, and protein kinase C signaling pathways. EGF receptor signaling involves in cell growth, angiogenesis and DNA repair. Deregulated and excessive expression of EGFR, the transmembrane receptor tyrosine kinase , is a feature and/or cause of a wide range of human cancers, especially in epithelium cell originated carcinomas. Blockade of EGF receptor signaling pathway represents a new perspective on the development of novel and selective anticancer therapies. Although considerable progress has been made in the application of EGF receptor—targeted antibodies and small molecule tyrosinekinase inhibitors, none of these agents is curative.Current therapies of its expression are still need to be improved. In RNA interference (RNAi), duplexes of 21-23 nt RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. It is approved that vectors containing shRNA expressing cassette could induce good result of RNAi. RNAi has shown enormous promise in gene silencing, the potential of which in developing new methods for cancer therapy is still need tested. In this study we want firstly to filtrate an effective RNAi sequence by reconstructing shRNA expressing plasmid wector ,using which to investigate the biological features of A431 cells(human epithelium tumor cell) testing the efficiency of RNAi in vitro. Then to establish a shRNA expressing adenovirus from the U6 expressing cassette to further confirm the effect of RNAi in HepG2 cells (cancer cell line of human heaptocarcinoma) in vitro . METHODS: 1. RT-PCR, Western Blot, FCM(flow cytometry ) and MTT were performed on the HepG2 cell line transfected by recombinant pSIREN-hERa to test the biology alterations of phenotype in HepG2 cells in vitro. pSIREN-hERs consisted of three 69 base-pair oligos coding small hairpin RNA containing 19bp of sense sequence and of antisense sequence which specifically targeting EGFR gene which were inserted into the downstream of U6 promoter in RNAi-Ready pSIREN-Shuttle Vector to reconstruct RNAi expressing recombinant plasmid vector for gene of EGFR ( recombinant pSIREN-hERs). 2. Transferred U6 shRNA expressing cassette from pSIREN-hERa to BD Adeno-X^TM Viral DNA to construct recombinant pAdeno-hER^RNAiDNA vector . Recombinant pAdeno-hER^RNAiDNA vector was packaged into HEK293 (human embryonic kidney) cells. Recombinant adenovirus ( Adeno-hER^RNAivirus) was harvested by lysing transfected cells. The presence of inserter expression cassette in recombinant adenovirus were verified by restriction analysis with Xho I digestion and PCR Analysis. HepG2 cells were infected by 100 MOI of recombinant virus for 72 hrs. Real-Time RT-PCR, Western-blot were applied to evaluate the efficiency of RNAi induced by recombinant virus.Results: 1. Sequencing analysis results demonstrated that 69 bp oligos were inserted into expected site and the insertion sequence was exactly correct in recombinant pSIREN-hER vectors. recombinant pAdeno-hER^RNAi DNA vector and recombinant Adeno-hER^RNAivirus were verified by restriction and PCR Analysis that they have aim gene. 2. The mRNA expressing level of erbB-1 was depressed with recombinant pSIREN-hERa by RT-PCR analysis compared with control group in HepG2 cells treated. Western-blot and FCNM of HepG2 cell treated with recombinant pSIREN-hERa indicated that the recombinant pSIREN-hERa decreased the level of EGFR protein obviously with cell proliferation reduced due to induction of apoptosis and cell cycle arrest. 3. The results of by using recombinant virus of 100 MOI to infect HepG2 cells showed that mRNA expressing level of erbB-1 was depressed by 77.85% compared with control group ; a similar result was observed by western-blot.CONCLUSION: siRNA-mediated inhibition of EGFR gene induced by shRNA expressing Adeno-hER^RNAivirus is capable of suppressing EGF receptor expression with significantly inhibiting cellular proliferation and tumor growth due to cell cycle arrest and apoptosis induced may constitute a useful approach in the treatment of human liver cancer and other EGFR over expressing carcinomas.