The Study Of Hrg-1 Gene Expression And Function

Prostate cancer (PCa) is one of the most common malignancy in men, which ranks the first and second place of cacer incidence and fatality respectively of men in the United States. In recent years, with the aging of population and the changing of lifestyles, the incidence of prostate cancer in China has been showing an increasing trend and becomes the third most malignant cancer of urological system, although it is lower than that of western countries. Moreover, prostate cancer has a high recurrence rate and there is no effective way to treat it. Therefore, the basic and applied studies of prostate cancer have been transferred from pure treatment to the combination of prevention and treatment, and the discovery and application of tumor markers will play important roles in screening high-risk groups, diagnosing cancer at earlier stage, identifying diagnosis and assessing prognosis. Currently, it has being more and more valued for the molecular researches of the development and treatment of protate cancer.HRG-1, a new gene found in preliminary studies, has a homology of 78% with DU-145 prostate cancer cell apoptosis related gene pcar. As an important member of the solute transporter family, HRG-1 had been thought as a hypothetical protein, and had not been formally nominated as HRG-1 (heme-responsive gene 1) until 2008. Human heme-responsive gene 1 (hHRG-1), whose GenBank accession number is 017842, locates on human chromosome 12q13.11 and is about 3.2Mb apart from the DMT1 (a gene encoding mammalian iron transporter). It is suggested by Bonifacino that HRG-1 protein has four transmembrane domains and a singal-sorting domain based of conserved tyrosine and acid-dileucine in the cytoplasmic carboxy terminus through the topology model and gene sequencing, In addition, residues situated in the C-terminal tail could potentially either directerly bind haem or interact with the haem side chain (FARKY) . However, it remains unknown for the function of HRG-1 and its relation with tumorigenesis. In the previous studies, we found for the first time that it was very different for the expression of HRG-1 mRNA in the tissues of prostate cancer, hyperplasia of prostate gland and tumor surrounding compared with normal tissues. There is also no systematic research about the distribution of HRG-1 in various human tumors, especially the expression and distribution in prostate cancer. Whether could HRG -1 gene become a new tumor marker and a drug therapy evaluation index for prostate cancer? In this study , we generated mice polyclonal antibody, which establish the foundation for further study about the expression spectra of HRG-1 in human tissues and its structure and function.Objective To obtain purified heme-responsive gene 1 (HRG-1 ) protein and get its polyclonal antibody. investigate its expression in tissues of difference human organs. And compare the differences of HRG-1 expression among the prostate cancer, benign prostatic hyperplasia and normal prostate tissue.Methods Encoding sequence for HRG-1 was amplified by RT-PCR from DU-145 prostate cancer cell line. Then it was inserted into PGEX-4T-1 vector. The recombinant vector was expressed in E. coliBL21 and the expressed fusion protein was purified. BALb/c mice were immunized with the protein to get the antiserum. The titers and specificity of antibodies were measured by ELISA and immunohistochemistry methods。The differences of HRG-1 expression were compared among the prostate cancer, benign prostatic hyperplasia and normal prostate tissue, Results The PGEX-4T-1- HRG-1 vector was constructed. Induced by IPTG, the fusion protein was expressed in E. coli BL21. The ELISA result indicated that the prepared polyclonal antibody had a high titerof 1∶13000。The expression of HRG-1 was investigated by immunohistochemistry in the prostate cancer, benign prostatic hyperplasia and normal prostate tissue. There were obvious differences between these 3 groups. Conclusion HRG-1 expressed pathologically high in prostate cancer tissue. Thus, it is expected to be a new tumor marker of the prostate cancer and the further structural and functional study will help to diagnose and treat prostate cancer.