Fundamental And Experimental Research On The Enhanced Expression Genes Located On Chromosome8in Prostate Cancer

Background and Objective:As a common malignant disease of the male reproductive system, prostate cancer (PCa) is now widely recognized as one of the toughest medical problems facing the male population. In the United States and Europe, PCa is known as a most common tumor and the cases have exceeded patients suffering from lung cancer and colorectal cancer. PCa has become NO.1killer to men’s health. On a global scale, PCa has a lower incidence in China. However, a conservative estimation shows that the incidence and the morbidity of PCa in China are rising. By2030, the incidence and the morbidity of PCa are predicted to be106.4%and111.4%respectively, with an average increase of4.8%and5.1%per annum. Statistics of Shanghai revealed that incidence of PCa in this city had risen from29/100,000men (standardized rate of24/100,000) in1991to1214/100,000men (standardized rate of678/100,000) in2001. So, in-depth study of the origination, development and lesion of PCa bear great clinical and scientific significance to prevention and treatment of PCa.The occurrence and development of PCa involved participation of multiple genes and stages. All along, it was considered that genome changes had accelerated the development and progression of tumors. In progression of PCa, this change had a bearing on the occurrence of tumor. Genetic factor was an important risk factor resulting in PCa. Therefore, genetic research of PCa helped to provide datas for study on pathogenesis of PCa and facilitates identification of specific PCa markers for the Chinese. Studies had shown that chromosome8was associated with PCa. Absence of the short arm of chromosome8Indicated early PCa and this was true to prostatic intraepithelial neoplasia and almost all PCa. However, in recent years, amplification of the long arm8q of the8chromosome, especially for8q21and8q23-24regions,was considered to have a bearing on formation and invasive progression of PCa, and it also indicated poor clinical PCa prognosis. To study on mutation of chromosome8genome, probe potential genes to mark PCa, and explore mutation of such genes and their mechanism on development of PCa and clinical diagnostic molecular target had important significance.Gene microarray also called DNA microarray, has a fingernail size, and its base surface could be divided into tens of thousands to millions of cells. Within any specified cell, a large number of nucleic acid molecules, with specific function and a length of about twenty nucleotide sequences, might be fixed. Since the formation of different arrays of immobilized molecular probes on the substrate, molecular detection of genetic materials could be achieved by molecular hybridization and parallel processing. Therefore, gene microarray could be used for genetic studies, forensic identification, disease detection and drug screening and so on. Being fast and with unparalleled efficiency and multi-parameter, gene microarray technology, as a great innovation, was major leap in terms of conventional biological techniques, such as detection, hybridization, genotyping and DNA sequencing technology. The Human Genome Project had identified so many genes, and expression of these genes could be detected by the microarray techniques. Development of these technologies offered new ways to fundamentally solve the problems of biomedical research. While the gene microarray could detect thousands of tumor-related genes one time. So the gene microarray might contribute a lots to formation, development and the metastatic mechanism of tumors.and it was promising in diagnosis and treatment of urinary tumors.Therefore, we selected abnormally upregulated genes on chromosome8in PCa and benign tissues by gene microarray, and further selected the upregulated genes on8q21-24by MALNO network,further more validated the overexpression of genes and proteins from cell and tissues experiments.In hope of finding new potential tumor markers about the incidence and progression of PCa on8q21-24by researching the relationship between the upregulated genes with clinical features and pathological mechanism of PCa.Materials1:The PCa and benign tissues for gene microarray testing and validation of alternative oncogenes were collected from patients in our hospital.PCa14cases.2:PC3/DU145/LNCaP cell lines and Rwpe-1were preserved by our hospital laboratory.3:PCa tissue microarray specimens for immunohistochemical experiment were purchased from Creative Bioarray in the United States.Cancer tissue100cases.Methods1:Four groups of PCa tissues and adjacent benign tissues were collected from hospitalized patients, and got purer cancer tissue and its adjacent tissues through LCM. Purified total RNA for microarray hybridization. Microarray datas analysis、and screening of over expressed genes by Agilent microarray. According to the microarray analysis and Agilent software analysis, the genes with expression more than1.5were considered to be over expressed genes. Made comparison with cancer tissue and adjacent tissues in chromosome8and screened the enhanced expression of genes.2:Analysised the biological behavior of the enhanced expression of genes on chromosome8by Panther nework. String analysis of screening protein interaction network graphs.3:Then, based on the analysis of MILANO website, input differentially expressed genes from chromosome8in the Primary Search Term box,8q21-24,and searched the whole database to get the final list of enhanced expression of genes.4:First,RT-qPCR was used to examine the expression status of candiated genes mRNA in10cases PCa and benign tissues,as well as the PC3/DU145/LNCaP cell lines.The second, RT-qPCR was used to examine the DNA copy number changes of the genes which were consistent with the mRNA Q-PCR in10cases of PCa and benign tissues,as well as PC3/DU145/LNCaP/RWPE-1cell lins.5:Western blot verified the different expression of alternative proteins in10cases of PCa and benign tissues.6:Performed immunohistochemistry to detect the differential expression of the candidate proteins from large sample tissues microarray.Statistical analysisStatistical software SPSS17.0was adopted for data analysis. Continuous variables were expressed as x±s. Performed t-test to the two independent samples to study the different expression of the mRNA and protein in PCa and the benign tissues. Used Pearson chi-square test to analyze immunohistochemical staining of protein in PCa.The P values less than0.05were considered to be statistically significant.Result1：Enhanced expression of GHLHE22, C8orf38, C8orf51, CTHRC1, DSCC1, E2F5, FAM49B, FAM86B2, hCG-19905, INTS8, LOC389641, MTFR1, MYC, OSGIN2, PAG1, PBK, POLB, TMEM65, TNFRSF19A, UBXN2B and ZMAT4were detected on chromosome8by gene microarray 2:The enhanced expression of genes on chromosome8were relates to various biological behaviors. E2F5and MYC genes had similar behaviors.Also, interaction between DSCC1and PBK protein、E2F5and MYC protein had been detected.3:The enhanced expression of genens located on8q21-24were CTHRC1, PAG1, FAM49B, TMEM65, MYC and E2F5.4:The overexpressions of PAG1、FAM49B、TMEM65、E2F5、MYC had been detected in10PCa and benign tissues(P<0.05).But,the higher were E2F5and MYC.Also, E2F5mRNA had the higher expression in all PCa cell lines,compaired with MYC,which had been found overexpressed in LNCap and Du145cell lines only(P<0.01). Expression of CTHRC1in PC3和DU145cells were equally higher than those in RWPE-1(P<0.01), Expression of PAG1and TMEM65inDU145and LNCap cells were equally higher than those in RWPE-1(P<0.01).5:DNA-qPCR analysis results showed that gene copy number changs of E2F5in PCa was significant higher than that in benign tissues (P<0.05). In PC3and LNCap cell lines, gene copy number changs was greater than normal RWPE-1(P<0.01), but in the DU145cell line there was no significant in gene copy number changs. MYC in PCa was significantly higher than that in benign tissue (P<0.01). Only in LNCap cell line was greater than RWPE-1(P<0.01).6:Western blot results showed that the expression of E2F5and MYC in PCa tissues were equally higher than those in benign tissue (P<0.001).7:Our immunohistochemical efforts to elucidate that expression of E2F5and MYC proteins was significantly increased (P<0.001) in PCa tissues. Moreover, over expression of E2F5was related to high clinical stage (P<0.001) and metastasis (P <0.001), while MYC over expression was related to positive metastasis (P< 0.001).Conclusion1:Gene microarray screening for PCa tumor marker was an effective method.2:PAG1、FAM49B1、TMEM65located on8q21-24had the relationship with PCa.3:E2F5and MYC were associated with metastasis and progression of PCa,4:E2F5、MYC genes copy number increased in PCa, indicated that these two genes played a role in the genetic pathogenesis of PCa.5:The biology behavior of molecular as well as clinical level in E2F5and MYC were consistent, which indicated that the synergistic effect of E2F5and MYC played an important role in inheritance and clinical progression. E2F5and MYC could be identified as a potential oncogene in PCa.