Searching And Analyzing Novel Prostate Cancer Ralated Genes Based On Mining SAGE Databases

Background Following on from the inspiring completion of the Human Genome Project, the Post-Genome Project (Functional Genomics) attracting more and more attention. The Post-Genome Project is a more detailed study of gene function mainly focused on transcriptomics and proteomics. It is a huge and hard work to completely annotate gene function for explaining the mechanism of life. For cancer Research, the information of cancer related gene's function is a key point in exploring the mechanism of carcinogenesis and cancer progression. Prostate cancer is one of the most common human malignant tumors and the second leading cause of cancer death in males of western countries. Nowadays, the number of prostate cancer patients is increasing in China because of the increasing age, changing life style and improving medical examination. Searching and analyzing genes differentially expressed between malignant and benign prostate tissues may be used to understand the molecular mechanism of prostate cancer and provide diagnostic markers or new targets for therapy. Progress in technology of high throughput analysis of global gene expression profile such as SAGE and cDNA microarray markedly accelerated the comparative gene-expression analysis and made many novel cancer related genes had been identified. Especially after the Cancer Genome Anatomy Project (CGAP) was established, more and more Internet-available SAGE databases and analysis tools is open to the public for in silico analysis of the large datasets about gene-expression profile, specially to compare computed gene expression profiles between cancer and normal tissues. Careful analysis in silico through these Internet-available tools might gave us many useful clues to search cancer related genes and further research. It will significantly reduce lab work and expense, though the results require the verification of biological experimental methods. So it is a wise strategy to search candidate prostate related genes by mining public database, verify it by biomedical experiments, study the function of interested genes and reveal the relationship with prostate cancer. It will benefit not only fundamental research about functional genomics but also clinical research of prostate cancer about novel diagnostic and therapeutic method.Methods After credible databases and rational statistical parameter were set up, SAGE digital gene expression displayer (DGED) were used to analyze differential-expression (>5fold) genes in malignant prostate tissues compared with normal prostate tissues. The SAGE tags were filtered by their confidence and our three criteria. Internet-available software and modified GLGI(Generation of longer cDNA fragments from SAGE tags for gene identification)were used to identify genes of confidential SAGE tags and the cDNA 3' end downstream of un-confident SAGE tags. The same strategy was used to got the genes specifically expressed in prostate compared with the other organs in males. Main functions of all candidate genes and their relations with prostate cancer were annotated through review of literatures and the interested genes for further research were be selected. For interested gene LOC389816, reverse transcription (RT)-polymerase chain reaction (PCR) was used to reveal the expression profile of LOC389816 in liver, heart, skeletal muscle, pancreas, colon, prostate, testis, lung, skin, stomach, kidney and spleen. Bioinformatical tools and methods were used to predict the function of LOC389816 gene by analyzing the sequence and the functional motif. For gene BRP44, 1 )Northern blotting was used to reveal the expression abundance in 10 prostate cancer samples and 8 prostate benign hyperplasia samples;2)Bioinformatical tools and methods were used to predict the function of BRP44 gene by analyzing the sequence and the functional motif. 3)The plasmid with fusion gene of BRP44 and green fluorescent protein(GFP) was constructed and LNCaP cells were transfected with the recombinant vector by liposome and observed subcellular localization of GFP-BRP44 and pure GFP by the Laser scanning Confocal microscope. 4)Down-regulation of BRP44 was accomplished by small interference RNA (siRNA) in LNCaP cells and the effects of down-regulation of BRP44 on cell proliferation and viability, cell cycle and the amount of secreted PSA were evaluated with the alamarBlue assay, flow cytometre and ELISA.Results: 1)By SAGE analysis, we got 28 up- and 27 down-regulated confident SAGE tags that respectively represent 55 genes. Of 55 genes, 23 genes have not been demonstrated related with prostate cancer and may be possible candidate related genes to prostate cancer. Meanwhile, got 13 genes specifically expressed in prostate. 9 of 13 genes have been demonstrated and 4 genes may be novel candidate genes. Only 2 of 30 un-confident SAGE tags got the longer cDNA sequences by modified GLGI and may be two novel genes or novel spliceosomes related to prostate cancer. 2)RT-PCR revealed that LOC389816 only expressed in prostate among 12 human normal tissues. Bioinformatics predicted information hinted that LOC389816 may be a membrane protein function as receptor of signal transduction. 3) Northern blotting revealed the expression abundance of BRP44 in 10 prostate cancer samples was higher than in 8 prostate benign hyperplasia samples and BRP44 was highly expressed in LNCaP and not expressed in PC-3 cell line. Bioinformatical predicted information hinted that BRP44 may be a regulatory factor which regulate some gene expression in cellular mitochondrion. LNCaP cells proliferation and viability significantly increased and the percentage of Synthesis(S) phase significantly increased and other phase of cell cycle decreased in 48 hours after down-regulation of 60% expression of BRP44. However, there was no significant change of the amount of PSA secreted by LNCaP cells.Conclusion: 1)In silico analysis of public SAGE databases by the Internet-available tools is a simple, effective approach to get prostate cancer related genes. It might provide useful clues for searching novel prostate cancer related genes. 2) LOC389816 is a gene which specifically expressed in prostate and possibly is a membrane protein which function as a receptor of signal transduction. It prospectively be a new diagnostic markers or new therapy targets for prostate cancer. 3) BRP44 is a up-regulated expression gene related with prostate cancer. The protein of BRP44 expressed in both nuclei and cytoplasm of LNCaP cell but the expression in cytoplasm was stronger than in nuclei. BRP44 possibly is a resolved protein regulated by oct-1 and revolved in regulating the gene expressing in cellular mitochondrion. BRP44 negatively regulated proliferation and growth of LNCap cell line, but had no key effect on PSA secreted pathway.