Expression Of Id1 In Hepatocellular Carcinoma And Its Relation With HBx

【Background】Primary liver carcinoma is one of the most common cancer in the world. About 90%-95% cases are hepatocellular carcinoma(HCC). Although a lot of progress has been made in the clinical research of HCC, the prognosis of HCC is still poor due to high incidence of recurrence and metastasis. HCC is the second leading cause of cancer motality in China. It has been proved that chronic hepatitis B virus (HBV) infection plays an important role in HCC development. HBV X gene is the shortest open reading frame in HBV genome that codes HBx. HBx play an important role in HBV infection and development of HCC. HBxAg and anti-HBx antibody can be detected in majority of liver tissues and sera of HCC patients. HBx is also a useful biomarker of HBV infection and predictor of HCC occurrence. HBx is an activator of transcription. It regulates apoptosis by influence mitochondria and many other factors such as caspases and survivin. HBx participates in many intracellular signal pathways,such as PI-3-K-Akt/PKB, Jak-STAT, RAS-RAF-MAPK signal cascade and it can also regulate cell cycle.Id(sInhibitor of Differentiation and/or DNA binding) are small members of helix-loop-helix proteins without the DNA binding domain. Ids act as dominant negative inhibitors of differentiation- specific bHLH transcription factors. Id proteins have four family members named Id1, Id2, Id3 and Id4. They promote the cell growth, differentiation, apoptosis and angiogenesis. Ids are able to promote cell proliferation and cell cycle progression through inactivation of tumor suppressor and activation of growth promoting pathways in mammalian cells. Evidence has strongly indicated Id1 as a positive regulator of cell growth and its expression may be a key factor required for tumor cell proliferation. So Id1 might serve as a useful predictor of HCC occurrence for patients with cirrhosis, and might be a potential target for therapy.【Objectives】(1) To detect expression of Id1 and HBx in HCC tissues by immuno-histochemical staining. (2) To investigate the relationship of Id1 expression with the clinicapathological characteristics of the tumors and the expression of HBx. (3) To construct two luciferase reporter gene vectors containing two Id1 promoter regions of different length. (4) To measure the Id1 promoter activity in HepG2 cells.【Methods】(1) Clinical tissue samples including HCC, liver metastasis cancer and hemangiomas were collected with detailed clinical data and pathological sections were made. (2) The expression of Id1 and HBx was detected in HCC tissues and other tissues. (3) Relevant factors influencing the expression of Id1 and HBx were valued. (4) Gene sequence of Id1 promoter region was searched on the net. The up- and down-stream primers of two different lengths of base pairs were designed, with appropriate restriction enzyme at cutting site added in them. (5) The genome of HepG2 cells was gained as PCR template. Id1 promoter gene was amplified by PCR method. (6) The promoters were inserted into luciferase reporter gene vector by ligation and the vector plasmid was gained and subjected to DNA sequencing. (7) HepG2 cell line were transiently transfected with luciferase reporter gene vectors containing two Id1 promoter regions with correct DNA sequence respectively (8) .Luciferase activity of cell lysis was measured and compared between the transfectants and the control group.【Results】(1) The double-positive expression rate of Id1 and HBx was 97.7% in HCC. (2) Id1 and HBx showed more intense expression in HCC samples than other benign samples. (3) HCC samples with lower differentiation showed more intense expression of Id1 and HBx. (4) Id1 and HBx were expressed at more intense degree in HCC samples with faster HBV amplification. (5) Serum AFP, ALT, AST, TBIL, PT, APTT, INR, FIB and tumor volume showed no relevance to the expression of Id1 and HBx. (6) Two Id1 promoters were successfully cloned, named Id1p1(1096bp) and Id1p2(266bp). (7) The target luciferase reporter gene vectors containing the two Id1 promoter regions were constructed successfully after further identification by DNA sequencing. (8) Id1p1/pGL3 and Id1p2/PGL3, both containing Id1 promoter core sequence, showed higher luciferase activity than the control group respectively.【Conclusion】(1) Id1 and HBx showed more intense expressions in HCC tissues than other benign tissues and they two had co-expression in HCC samples. (2) The incidence of Id1 and HBx expressions and co-expression were higher in samples with poorer difference and higher HBV replication rate. Serum AFP, ALT, AST, TBIL, PT, APTT, INR, FIB and tumor volumes showed no relevance to the expressions of Id1 and HBx. (3) Two target luciferase reporter gene vectors containing Id1 promoter region were constructed respectively and successfully transfected into HepG2 cell line. (4) Id1p1/pGL3 and Id1p2/PGL3 both showed increased luciferase activities compared with the control group.