Overexpressed Id-1 Is Associated With Patient Prognosis And HBx Expression In Hepatitis B Virus-related Hepatocellular Carcinoma

【Background】Hepatocellular carcinoma (HCC) is one of the most common malignant tumors world wide. In Asia-Pacific countries, especially in China, the main cause of HCC is chronic hepatitis B virus (HBV) infection. Early diagnosis is critical for better treatments and improved clinical outcomes. Patients with early-stage HCC can be treated using curative treatments, such as resection or liver transplantation. Nowadays, no more than 30% of HCC patients can be early diagnosed and received appropriate treatment. More patients with advanced HCC have much worse outcomes because of the delayed treatment. So, more studies focused on whether there are novel biomarkers that predict the risk of tumorigenesis and the survival time for large groups of HBV-infected patients are needed.HBV plays an important role in HCC carcinogenesis. In China mainland, over 80% HCC patients are accompanied with chronic HBV infection. Moreover, the lifetime risk of HCC has been shown to be increased even in patients with occult HBV infection and after hepatitis B surface antigen (HBsAg) clearance. HBV could be integrated into the host genome to promote the carcinogenesis process by either directly or indirectly pathways. Hepatitis B virus X protein (HBx), one of the HBV-encoded proteins, accounts for the oncogenic properties of HBV in liver cells. In the pathogenesis of HBV-associated HCC cells, HBx may play a critical role by inducing progressive changes and facilitating the development of carcinoma. HBx could regulate different signaling pathways through interaction with a variety of proteins, such as the p53, DDB1, Caspase3 and proteasome subunit. HBxAg and anti-HBx are both over-expressed in the serum of HCC patients and tumor tissues.Id proteins (Inhibitor of Differentiation and/or DNA-binding Protein) lack a basic domain and serve to inhibit the DNA binding of basic helix-loop-helix (bHLH) transcription factors by heterodimerization with bHLH proteins. As a member of the Id protein family, Id-1 plays a negative role in cell differentiation. The Id-1 gene has been shown to be highly expressed in a variety of primary human tumors including HCC. There is increasing evidence showing that deregulated Id-1 expression may contribute to various properties of tumor cells, including induction of aberrant cell proliferation, inhibition of differentiation, stimulation of angiogenesis and induction of genomic instability. Furthermore, the Id-1 expression level is often found to be correlated with malignant tumor phenotype, dedifferentiation and poor clinical outcomes of patients. Over-expressed Id-1 could induce proliferation of HCC cells and it may serve as a useful marker for determining HCC risk in patients with cirrhosis. So it is meaningful to further elucidate the role of Id-1 in HBV-related HCC.【Objectives】(1) To detect the expression of Id-1 and HBx in HBV-related HCC tissue samples. (2) To analyzed the correlation between Id-1 and HBx expression levels and clinicopathological features of patients. (3) To detect whether Id-1 and HBx are co-localized in HCC cells. (4) To investigate the effect of HBx on Id-1 expression in HCC cell line. (5) To construct the lentiviral vector with Id-1 siRNA. (6) To measure the silent efficiency of lentiviral vector with Id-1 siRNA after infecting the HepG2 cells.【Methods】(1) Tumor tissue samples obtained from a total of 96 HCC patients. The expression of Id-1 and HBx proteins of these samples are detected by immunohistochemical (IHC) staining and evaluated by two independent pathologists. (2) The corrections between the clinical pathological parameters and the IHC scores for Id-1 or HBx and the prognostic significance were statistical analysesed by SPSS software. (3) Immunofluorescent staining was used to detect the colocalization of Id-1 and HBx proteins in HCC cells. (4) The mRNA and protein expression of Id-1 in HCC cell lines (HepG2, HepG2.2.15, SMMC7721 and FHCC98) and hepatic cell line (HL7702) are detected and compared by Realtime-PCR and Western Blot. (5) The mRNA and protein expression of Id-1 in HepG2-X (stably transfected with HBx) and HepG2-PC (transfect with empty vector) are detected by Realtime-PCR and Western Blot. (6) Luciferase reporter gene vector with Id-1 promoter was used to investigate whether epitopic expression of HBx could stimulate higher luciferase activity of Id-1 promoters in HepG2-X cells. (7) Construct the lentiviral vector with Id-1 siRNA using the most effective Id-1 RNA interference sequence. (8) Measure the silent efficiency of lentiviral vector with Id-1 siRNA after infecting the HepG2 cells by Realtime-PCR and Western Blot.【Results】(1) Over-expression of Id-1 and HBx were found in 64.6% and 74.0% of HBV-related HCC specimens, respectively. The expression of Id-1 was positively correlated to that of HBx. (2) Over-expression of Id-1 was correlated with the histological grade, portal vein invasion, lymph node metastasis, HBsAg and Child-Pugh classification. Patients with Id-1 overexpression had both shorter disease-free and overall survival times. (3) Colocalization of Id-1 and HBx was found by paired IHC and confocal study. They were both expressed in the cytoplasm and/or the nucleolus in HCC cells. (4) The expression of Id-1 mRNA and protein were both higher in HCC cell lines (HepG2, HepG2.2.15, SMMC7721 and FHCC98) than they were in hepatic cell line (HL7702) by Realtime-PCR and Western Blot. (5) HepG2-X showed a significantly higher Id-1 expression compared to HepG2-PC cells transfected with empty pcNDA3.1 plasmids, suggesting that HBx may up-regulate Id-1 in HCC cells as shown by real-time PCR and western blot. (6) Epitopic expression of HBx could stimulate higher luciferase activity of Id-1 promoters in HepG2-X cells by luciferase reporter gene system. (7) We successfully constructed the lentiviral vector with Id-1 siRNA named Id-1-RNAi-LV. The virus titer is 2.0×10E9 TU/mL. Id-1 mRNA and protein expression were significantly down-regulated after infected by Id-1-RNAi-LV in HepG2 cells. The silent ratio is 61.2%.【Conclusion】(1) Id-1 overexpression was correlated with HBx expression, they were both colocolized in HCC cells of HBV-related HCC patients. (2) High expression of Id-1 were correlated with malignant HCC tumor phenotype such as serum HBsAg, histological grade, lymph node metastasis, portal vein invasion, Child-Pugh classification and poor clinical outcomes in HBV-related HCC. (3) Id-1 expression was higher in HCC cell lines than hepatic cell line. (4) Epitopic expression of HBx could stimulate Id-1 promoter. (5) Lentiviral vector with Id-1 siRNA was successfully constructed and have significantly silent efficiency in HepG2 cells. (6)Thus, Id-1 may serve as a useful prognosis marker for HBV-related HCC patients, and Id-1 overexpression in HBV-related HCC may be partially attributed to the effect of HBx. The lentiviral vector with Id-1 siRNA can be used for further researches on the mechanism of Id-1 involved in hepatocellular carcinoma and may act as a noval molecular targeting therapy of HBV-related HCC.