The Expression And Role Of Anti-proliferation Gene BTG2^PC3/TIS21 In Hepatocellular Carcinoma

Background and objective In China, Hepatocellular carcinoma is one of the most common malignant tumor with low surgical resection and high recurrence rate, which occur latently, invase and metastasize early. Meanwhile, it is not sensitive to radiotherapy and chemotherapy, the therapeutic efficacy of liver transplantation, immunotherapy and interventional therapy is also not ideal. Nowadays, molecular biology develops rapidly, with more and more molecular mechanism on hepatic carcinoma are uncovered,molecule target therapy becomes a hopeful therapeusis to improve patient outcome. Consequently,the study of molecular machanism of signal transduction in hepatic carcinoma becomes a key point for large molecular therapy. With investigating deeply on the interaction of signal protein molecule concerned with occurrence,adhesiveness,invasiveness,diffusion and metastasis of hepatic carcinoma,it becomes meaning for us to illuminate the molecule mechanism of hepatic carcinoma and discover the candidate molecular target of the capital signal protein.BTG2PC3/TIS21,antiproliferative gene, has been known as one of the early growth response genes and belong to the BTG /Tob anti-proliferative protein family.This ability of BTG2PC3/TIS21 to inhibit the cell cycle might have a functional role in the growth arrest triggered by a variety of stimuli, such as DNA damage or other types of cellular stress, cellular differentiation, and apoptosis. Meanwhile BTG2PC3/TIS21 encoding protein is a secreted protein with secreted peptide. It has a close relationship with some anti-multiplication related inhibiting cancer reaction genes such as P53,P73 and RB.But the conclusion of BTG2PC3/TIS21 is a tumor suppressor is not clear. Therefore,it is an important chance for us to reveal the expression and role of BTG2PC3/TIS21 in hepatocellular carcinoma.In our previous study on BTG2PC3/TIS21 gene expression in hepatocyctes,we found that BTG2PC3/TIS21gene was overexpression in investigation of hepatocyte proliferation different by RDA in the rat model of 2/3 hepatectomy,these results suggested that expression of BTG2PC3/TIS21 gene probably participated in regulation of liver regeneration and was an immediate early gene that was closely related to liver regeneration and hepatocarcinogenesis. It was the first report about the relation between BTG2PC3/TIS21 and liver regeneration.Because the correlation between BTG2PC3/TIS21 and hepatocellular carcinoma were not clear,we induced hepatocellular carcinoma in rats by administering DEN orally in their drinking water, and observed the changes of BTG2PC3/TIS21, P53, and cyclin D1 at mRNA and protein levels over the observation period.In addition,we have characterized the expression of BTG2PC3/TIS21mRNA in HCC tissues,surrounding liver normal tissues by high-flux tissue microarray technique combined with in stiu hybridizayion, and analysis the expression of P53 ,cyclinD1 and cyclinE in HCC patients whose expression of BTG2PC3/TIS21 mRNA were absent by immunohistochemistry. We also evaluated the expression of BTG2PC3/TIS21 in a large cohort of human hepatocellular carcinoma of various histologic types,its relationship to clinicopathologic variables and P53,cyclin D1,cyclinE overexpression to enrich and consummate the molecular mechanism of BTG2PC3/TIS21 is a tumor suppressor in hepatoma cell.Methods1. Modified Diethylnitrosamine-induced primary hepatocellular carcinoma rat model was established.The pathological changes and ultrastructural morphology of liver tissues were studied by gross observation,histologic examination and electron microscopy.2. By the method of RT-PCR and Western Blot, to detect the mRNA and protein expression of BTG2PC3/TIS21,P53, and cyclin D1 from liver tissues of rat model.3. The expression of BTG2PC3/TIS21 mRNA of fetal liver and part of liver cancer cell lines was measured by RT-PCR and Northern blot analysis.4. Determined the expression and location of BTG2PC3/TIS21 mRNA on tissue microarray (TMA) of HCC via in situ hybridization (ISH).5. Immunohistochemistry demonstrated that BTG2PC3/TIS21 and P53, cyclin D1,cyclinE were correlated with a series of clinico-pathologically relevant parameters (Tumor size, Differentiation degree and HBsAg, Metastasis) in TMA of HCC.Results1. Twenty-four Wistar rats (96%) hepatocarcinoma model were set up successfully after 16 weeks.The procession of hepatocarcinogenesis in this model consisted of three stages (hepatictoxic lesion , hepatic proliferation/ cirrhosis , hepatic carcinogenesis). A series of injured alterations were found in the mitochondria, rough endoplasmic reticulum, smoth endoplasmic reticulum and the nuclei of the hepatocytes during the hepatocarcinogenesis. we also observed that hepatocytes phagocytized other cells during the process of hepatocarcinogenesis.2. The BTG2PC3/TIS21 mRNA was overexpressive during the early stage after DEN treatment,the expression level was peaked at 5 weeks and then it gradually decreased after 16 weeks; we did not observe the expression of BTG2PC3/TIS21 mRNA in normal rat liver. A significant increase in P53 was found in 5th week after the administration of DEN,then it gradually decreased after 16 weeks;Loss of expression of cyclin D1 protein in the early stage after DEN treatment, and its expression was peaked at 16 weeks.3. The BTG2PC3/TIS21 protein was overexpressive during the early stage after DEN treatment,the expression level was peaked at 5 weeks and then it gradually decreased to the normal level after 16 weeks; A significant increase in P53 was not found until 5th week after the administration of DEN,then it gradually decreased after 16 weeks;The expression of cyclin D1 protein was increased significantly according to the progression of tumor, and was peaked at 16 weeks and 5 weeks respectively.4. The BTG2PC3/TIS21 mRNA was overexpressed in fetal liver,7701and 7721 hepatoma carcinoma cell lines,but decreased in HepG2.By using in stiu hybridizayion in HCC tissue microarray, we observed that BTG2PC3/TIS21 mRNA expressed in 71.19% HCC and localized predominantly in the cytoplasm of tumour cells. 82.35% P53,94.12% cyclinD1 protein and 76.47% cyclinE protein expressed respectively in HCC patients whose expression of BTG2PC3/TIS21 mRNA was deficient.Expression analysis of BTG2PC3/TIS21 protein reavealed the loss of nuclear expression in 49.2%(32/65) HCC tissues. The expression of both BTG2PC3/TIS21 mRNA and protein related to increasing tumor grade significantly (P<0.05).Conclusion1. The expression of BTG2PC3/TIS21 increased significantly duing the early formation process of hepatoma(acute injury period) and decreased during the late stage of the cancer,and its expression is restricted in space and time to areas.2. The expression of BTG2PC3/TIS21 may be in coordination with P53 and be a factor involved in down-regulating cyclinD1 expression during hepatocarcinogenesis.3. Loss of nuclear expression of BTG2PC3/TIS21 may be related to the carcinogenesis and progression of HCC.4. BTG2PC3/TIS21 expression in HCC may regulate through P53-dependent and -independent mechanisms.5. BTG2PC3/TIS21 may be a factor involved in down-regulating cyclinD1 and cyclinE expression in the progression of hepatocarcinogenesis.