Identification Of Differentially Expressed Genes In Prostate Cancer Mouse Model And Function Analysis Of TRIM59 Gene

Prostate cancer (PCa) is the most frequently diagnosed cancer for men. PCa has become the second leading cause of death in male malignant carcinomas. More significantly, the incidence of PCa has been increasing in recent years among Chinese. Simulated development of PCa cells and animal models are commonly used in preclinical research on PCa studies. A knock-in mouse adenocarcinoma prostate model (KIMAP) at a locus of PSP94 (prostate secretory protein of 94 amino acids) gene was therefore established. The KIMAP showed kinetics of tumor development which is close to human conditions. Considering the reasons mentioned above, the concerned study was carried out based on the following components: (1) Microarray used to screen the differential highly expressed genes with unknown function in prostate between wild type mice and KIMAP prostate cancer mouse model (by 20th week and 60th week). The RT-PCR analysis and RT-qPCR analysis were used to validate the result of screening. (2) The coding sequence of mouse gene with unknown function was cloned by RT-PCR from total RNA of the mouse prostate. The structural domain, putative signal sequences, transmembrane domain, and secondary & tertiary structure of protein were predicted by bioinformatics. (3) The prokaryotic expression vector was constructed and expressed in prokaryotic cells. The purified protein was obtained via affinity chromatography. And the recombination protein was detected by Western blotting. (4) The purified fusion protein was used as immunogen for immunizing the rabbits. (5) The Gene functions were studied with polyclonal antibody. Results listed as following:(1) Screening the expression of KIMAP mouse model related genes by microarray. The results indicated that 491 genes with their expression up-regulated more than 2-fold from 20th week KIMAP mouse comparing to the wild type mouse; 442 genes up-regulated more than 2-fold from 60th week KIMAP mouse comparing to the wild type mouse. There were 72 genes which up-regulated more than 10-fold in KIMAP mouse model comparing to the wild type mouse. The function of 67 genes were known in highly expressed genes (72 genes), and the function of 5 genes (72 genes) were unknown. The genes of unknown function were verified by RT-PCR results and RT-qPCR results.(2) The whole coding sequence (1212 bp) of TRIM59 gene was gained, which encoded a polypeptide of 403 amino acids. The homologies of cDNA nucleotide sequence of TRIM59 (NM₀25863) with the counterparts of human (NM₁73084) and chick (NM₀01031320) were 85% and 72% respectively, and the homologies of peptide sequence were 86% and 75% respectively. TRIM59 was a mono transmembrane protein and the transmembrane domain was located at 329-348 peptides. The TRIM59 protein included 3 functional domains of RING (10-60 peptides), B-box (92-134 peptides) and transmembrane (329-348 peptides).(3) We got the fragment of mouse TRIM59 with the coding region (961-1338 bp) by RT-PCR. Then the target fragment was cloned into Prokaryotic expression vector pGEX-2T using the recombinant DNA technique and it was confirmed by DNA sequence analysis and restriction enzyme digestion. After induced by IPTG, the fusion protein was expressed in E.coli BL21 at 45 kDa. The fusion protein was purified and collected via affinity chromatography(4) We immunized rabbits with purified fusion protein (GST-TRIM59). The rabbits were injected with purified fusion protein as immunogen; we collected the immune sera of those rabbits. The sera separated from the blood and the antibody (IgG) separated from the immune sera were purified by Protein A agrose. Those antibodies titers and specifies detected by ELISA and Western blotting. Indirect immunofluorescence analysis showed that TRIM59 located in the cytoplasma of NIH3T3, indicating as the possible main function place for TRIM59. TRIM59 highly expressed in prostate cancer cells which was detected by Western blotting and IHC.(5) Our study demonstrated that E3 ubiquitin ligase activity was a novel property of TRIM59. It was found that TRIM59 underwent self-ubiquitylation in vitro when combined with the E2 enzyme UbcH5a and UbcH5c.The ubiquitylation was dependent on its RING finger domain. Further evidences showed that TRIM59 could also be self-ubiquitylated in vivo. Our data showed that Cys was an important amino-acid residue of RING domain to keep E3 ubiquitin ligase.In this research, prostate cancer-related genes of unknown function have been studied via microarray screening and functional study within prostate cancer mouse model. It lays the theoretical foundation for identifying new therapeutic targets and diagnostic markers for prostate cancer.