Prostate Cancer Gene Chip Study And Cry61 Gene Promoter Region Of Genetic Variation And Genetic Susceptibility To Prostate Cancer Research

Background:Prostate cancer (PCa) is the most common malignant urological tumor in men. In 2009, it is estimated that 192,280 new cases of PCa are expected and 27,360 deaths will occur in the USA. It is the leading incidence of all malignant tumor and second highest mortality after lung and bronchus tumors in the USA.In China, as lifestyle are becoming more westernized, the mortality of PCa increases from 1.5/100,000 in 2000 to 1.7/100,000 in 2005. The increasing incidence and mortality not only endange the health of patients, but bring great burdern to family and whole society. Many factors are associated with PCa, such as age, racial backgrounds, environmental factors, genetic background and sex steroid hormone levels. Only a small portion of people with risk factors develop into prostate cancer, which reveals the difference of individulized susceptibility. The prostatic tissues with bio-activity provide the base of molecular biological research. Most of tumor tissues, as called cancer nodule, locate within the prostate and they can not be observed during prostatectomy regardless of open approach or laparoscopic approach. Even it is quite difficult to differentiate with normal prostatic tissue when the gland is incised. As a result, the establisment of prostatic tissue bank becomes more inconviennce than other urological tumor. The recent reseach reveal CYR61 gene participates in the development and progression of various cancers.Elevated expression of CYR61 has been detected in the invasive breast cancer cell lines, pancreatic cancers and paediatric tumors.Objective:1. To establish the tissue bank of prostate cancer.2. To study the expression difference between prosate cancer and benign prostatic hyperplasia, open prosatectomy and laparoscopic prostatectomy using DNA microarray.3. To study the association between this polymorphism and PCa risk and the expression of CYR61 with different genotypes in PCa tissues in the Chinese populations.Methods:1. Establishment of tissue bank of prostate cancer. We cooperate with urology department of John Hopkins universtiy. We take an effective protocal of preserving prostate cancer tissue with the help of pathologist in our hospital. The clinical data of each sample is collected as well as epidemological investigation and postoperative follow-up. Detailed information of investigation includes genearl condition, personal history, past history, tobacco use, alcohol use, family history of cancer, exposure to risk factors, clinical data (serum PSA level, Gleason score, pathological results, staging, metastasis etc.). All informations are kept in data base in charged by an appointed doctor. Each subjects donated 5 mL of blood for genomic DNA extraction. Genomic DNA was isolated and purified from leukocytes and stored at-20℃for genotyping. Until now we have over 100 cases of prosatic cancer tissue and RNA from some samples have been confirmed well preserved through frozen section, HE staining and RNA extraction.2. Analysis of human whole genome expression microarray. A total of 26 RNA samples were profiled, including samples from 8 LRP cases,17 ORP cases, and 1 reference RNA sample previously prepared from a transurethral resection of the prostate (TURP) specimen of benign prostatic hyperplasis (BPH). We generated genome-wide expression profile for each of the 26 samples using the routine 2-color design and a total of 44,000 genes/probes were used for the gene expression profile.Expression profiles for each samples were normalized independently, using the standard locally weighted least squares regression (LOWESS) procedure. Volcano plot analysis was performed using the GeneSpring software (Agilent Technologies). To determine the expression differences between PCa and BPH, LRP and ORP cases, we calculated the absolute fold change (FC) value and p values.3. Analysis of the association between this polymorphism and PCa risk. Polymorphism was determined by using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. mRNA from prostatic tissue with different genotype was measured by both reverse transcription-PCR (RT-PCR) and real-time quantitative RT-PCR.Results:1. We now have 141 tissue samples of prosate cancer and over 835 blood samples. Each sample is attached with detailed clinical data and regular follow-up. All informations are stored in data base. RNA from part tissue sample was extracted and confirmed well preserved through clear 28s and 18s band under gel electrophoresis.2 75 genes/probes were identified as candidate differentially expressed genes between ORP and LRP cases.Among the 75 genes/probes,67 demonstrated higher expression in ORP cases. Another striking feature is that top-ranked genes, including FOSB, IER2, JUN, ATF3, FOS, are known to be involved in acute stress response.House-keeping genes commonly used as control genes in quantitative RT-PCR analysis did not show differential expression. These house-keeping genes included GAPDH, HPRT1, and TFRC, that were previously used to assess expression differences caused by surgical manipulation. CYR61 has a high expression in prostate cancer.Conclusions:1. We successfully established tissue bank of human prostate cancer. The effect of the LRP procedure on RNA quality and genome-wide transcript levels is minimal, qualifying LRP specimens collected post-operatively for routine molecular analysis.Blunted stress response in LRP specimens is manifested in the reduced expression of stress response genes in these specimens, and may be mediated by CO2 insufflation in LRP.CYR61 polymorphism has an effect on risk of PCa. The variant TT genotype was associated with an increased PCa risk and increased expression of mRNA of CYR61 compared with GG or GG/GT genotype.