The Expression And Trinucleotide Repeats Of Androgen Receptor In Chronic Hepatitis B Virus Associated Liver Diseases

It is known that about half of the global patients with chronic hepatitis B virus (HBV) infection are in China. More efforts have focused on the basic study, diagnosis and therapy of the chronic hepatitis B virus. Because chronic HBV-associated liver diseases, especially hepatocellular carcinoma (HCC) is the male predominance, the role of androgen receptor (AR) and its pathway became the focus of the study. Thus, some researchers proposed that certain HBV factors cooperated with AR signaling pathway in HBV-associated hepatocarcinogenesis. To date, the expression of AR in HCC or normal liver is inconsistent, and still controversial in independent studies. Majority of the early studies employed indirect binding assays to detect AR in the liver tissues, not direct antibody reaction. Any more, there were differences of the quality among antibodies by different methods. More precise quantitative methods of direct detection of AR protein are needed. The more important is that the most of previous studies were small-cases studies, and not considering the etiological differences in individual backgrounds. Evaluation of AR status due to different etiological factors in large cases is required for critical understanding of its role in HCC pathogenesis. In hepatocyte cell line and hepatitis B virus x gene (HBx) transgenic mice, HBx has been documented to enhance the transcriptional activity of the AR. In vitro, HBx also increases the AR protein level and possibly influences the stability of AR by proteosome activity. However, the relationship between HBx and AR has not yet been demonstrated in the clinical samples.There are two trinucleotide repeats in the first extron of AR gene. In vitro, the numbers of CAG and GGC correlated with the expression and transcriptional activity of AR. The clinical studies to prostate cancer revealed that the number of CGA and GGC repeats correlated with the risk of prostate cancer, the grade of the tumor, progress and metastasis in patients. In addition, the length of CGA and GGC repeats was reported in a few documents to correlate with the risk of HCC in liver. Today, the relationship between the length of CGA and GGC repeats and chronic HBV-associated liver diseases is not clear. Whether the number of CGA and GGC repeats is related with the prognosis of patients with chronic hepatitis B is unknown. Chronic HBV-associated liver diseases (especially chronic hepatits B and HBV-associated HCC) as material, we detected the AR and HBx expression to find the relationship between them in the present study, and to investigate the methylation regulation for AR expression. We also detect the number of CGA and GGC repeats in chronic HBV-associated liver diseases, to find out the relationship between them and the prognosis in patients with chronic hepatitis B.Part I The epidemiological characteristics of chronic HBV infectionPurpose:To detect the epidemiological characteristics of chronic HBV infection and relationship between HBV and hepatocellular carcinoma. To determine the incidence and distribution of HCC in the world and find the main risk factors, or the aim and significance of the study.Methods:To revive papers in the past ten years, about epidemiology, etiology and pathogenesis of HBV infection and HCC.Results:The frequency of HBV infection is very high in the world, especially in Asia, Africa, South Europe and Latin America. Half of the patients with chronic HBV infection are in China. It has been documented that hepatocarcinogenesis is related with HBV infection obviously. Compared with the frequency of hepatocarcinogenesis in the normal controls without HBV infection, the risk of HCC is increased by 1000 folds in the patients with cirrhosis induced by HBV infection. One of the serious prognoses of HBV infection is HCC. HCC is most HBV related in China. The positive HBV indication is found in about 90% HCC cases.Conclusions:A remarkable feature of HBV-associated HCC is male predominance, and male is also one of the important risk factors of HB V-related hepatocarcinogenesis. It has been suspected to be the most important viral factor in hepatocarcinogenesis. However, the relationship between HBx and AR has not yet been demonstrated in the clinical samples. There are CGA and GGC repeats in the first extron of AR. Except for HCC, few documents report the distribution of these repeats and the relationship between them and liver disease. It is still unclear that the number of the CGA and GGC repeats is involved with the prognosis of patients with chronic HBV infection. Part II The expression of androgen receptor in chronic HBV-associated liver diseasesPurpose:83 HBV-associated HCC and 189 chronic hepatitis B cases as materials, we detected the AR and HBx expression in both mRNA levels and protein levels, and to find the relationship among them and clinical characteristics in the present study. Compared with the negative controls, we tried to find the role of AR in HBV-associated hepatocarcinogenesis.Methods:Eighty-three cases of surgically resected HBV-associated HCC between 2007 and 2008 were selected from Liver Cancer Institute at Zhongshan Hospital. Meanwhile,14 cases of the HBV-negative HCC surgically resected were recruited as negative control, and 13 peritumoral liver tissues from hemangioma surgically resected served as benign control. One hundred and eighty-nine liver biopsy samples from Department of Pathology, Fudan University between 1988 and 2002 were also selected for this study. Meanwhile,75 cases whose diagnosis as "No pathological diagnostic abnormal" were chosen as negative control for liver biopsy samples. The expressions at mRNA levels were detected by fluorescence quantitative real-time RT-PCR. Western blot investigated the protein expressions. The expressions in tissues were showed by En Vision immunohistochemical stain.Results:In 83 HBV-associated HCC cases, in both peritumoral tissues (P=0.000) and tumors (P=0.000), AR mRNA levels correlated positively with HBx; The expression of AR (P=0.011) and HBx (P=0.000) was significantly higher in peritumoral tissues than in tumors; HBV-associated HCC cases had significantly higher AR mRNA than HBV-negative HCC in both peritumoral tissues (P=0.027) and tumors (P=0.021). By Western blot analysis, in both peritumoral tissues (P=0.008) and tumors (P=0.012), the expression of AR correlated positively with HBx in HBV-associated HCC cases; The expression of AR (P=0.018) was significantly higher in peritumoral tissues than in tumors; HBV-associated HCC cases had significantly higher AR protein than HBV-negative HCC (P=0.029) and benign control (P=0.029) in peritumoral tissues. Immunohistochemical stain showed that the expression of AR correlated positively with HBx (P=0.049) in peritumoral tissues. The expression of AR (P=0.038) was significantly higher in peritumoral tissues than in tumors; HBV-associated HCC cases had significantly higher AR protein than HBV-negative HCC (P=0.021) and benign control (P=0.043) in peritumoral tissues. In liver biopsy cases, HBV infection changed the normal fluctuation of AR with age. Not only at mRNA levels (P=0.046) but also at protein levels (P=0.049), the AR expression in peritumoral tissues correlated significantly with the tumor differentiation. In chronic hepatitis B patients, the expression of AR correlated with the grade of Scheuer scores (P=0.034).Conclusions:To clarified the expression of AR in HBV-associated HCC and chronic hepatitis B cases. AR plays a role in HBV-associated hepatocarcinogenesis. HBx-induce AR expression may be a mechanism of hepatocarcinogenesis. AR had stronger expression in peritumoral tissues than in tumors implied that the expression of AR increases during preneoplastic stages and that progression towards cancer development can suppress maintain AR expression levels. It is therefore proposed that androgen therapy may be ineffective after establishment of tumor. In chronic hepatitis B, AR pathway may be related with inflammation.PartHI The trinucleotide repeats of androgen receptor and the prognosis in patients with chronic hepatitis BPurpose:To detect the numbers of CGA and GGC repeats in the first extron of AR in HBV-associated HCC and chronic hepatitis B cases. To clarify the relationship between the numbers of CGA and GGC repeats and prognosis in patients with chronic hepatitis B, by a retrospective study.Methods:All of the 145 biopsy cases of adult patients were followed up for two to eighteen years and classified into two groups at first:Cirrhosis and Non-Cirrhosis. The Non-Cirrhosis was still divided into ALT Fluctuation and ALT Normalization. Eighty-three cases of surgically resected HBV-associated HCC and 53 adult cases of liver biopsy samples whose diagnosis as "No pathological diagnostic abnormal" were also chosen as materials. DNA was prepared from formalin-fixed and paraffin-embedded samples. CAG and GGC repeats were amplified by PCR with fluorescence primers. The products were then analyzed by genescan and calculated the numbers of the repeats.Results:In HBV-associated HCC cases, the mutations of CAG and GGC repeats presented in tumors. CAG repeats correlated with serum HBeAg (P=0.003) and HBeAb (P=0.032). The patients infected with HBV of genotype C had longer CAG repeats than those infected with HBV of genotype B (P=0.041). There was no significance for CAG and GGC repeats between ALT Fluctuation and ALT Normalization groups, and between Cirrhosis and Non-Cirrhosis groups. The CAG and GGC repeats in male patients had no differences among chronic hepatitis B, HBV-associated HCC and negative control patients. In female, the CAG repeats in chronic hepatitis B patients was shorted obviously than that in IIBV-associated HCC patients (P=0.037). Nineteen repeats of CAG as cutoff point, the frequency of more than 19 repeats in HBV-associated HCC patients was higher than the frequency in chronic hepatits B patients (x2=5.71, p=0.018).Conclusion:In HBV-associated HCC patients, longer CAG repeats are companied with high frequency of HBeAg and low frequency of HBeAb in serum. CAG and GGC repeats have no correlations with serum ALT levels and cirrhosis progress in patients with chronic hepatitis B. In male patients, the CAG and GGC repeats do not affecte the risk of cirrhosis and HCC. In female patients with chronic hepatitis B, longer CAG repeats increase the risk of HCC. Those with the average number of CAG repeats more than 19 have more frequency of HBV-associated hepatocellular carcinogenesis.