The Proteomics-based Screening Of Stromal Marker In Prostate Cancer And The Clinical Study Of MYL9 Expression

Background and Objective:Prostate cancer is one of the most common male malignant urologic neoplasms, and is a threat to the health of elderly men. It is the second common male tumor around the world, and it has the sixth highest tumor relative mortality. There is great difference of prostate cancer incidence and mortality among different districts. In USA, it has the highest cancer incidence and mortality. Meanwhile in Asian countries, the incidence of prostate cancer is still in a low level, but with the population aging and the development of diagnostic methods, the incidence has grown up during recent years, and it is the third common cancer in male genitourinary system cancer, following bladder cancer and renal cancer. Now, the regular tests for screening and diagnosing prostate cancer include the detection of serum PSA level and prostate biopsy. However, the PSA level is not sensitive and specific enough, not suitable for differing advanced and indolent cancer, contributing a result of unnecessary overtreatment. In order to diagnose prostate cancer early, and make personal treatment for prostate cancer, it is necessary to find more efficient biomarker to decrease the mortality and improve life quality.It is not completely clear about the cause and pathogenesis of prostate cancer. Most study about the pathogenesis of prostate cancer mainly focused on the prostate epithelial cell in the past years. It is believed that the initial cause of prostate cancer is the gene mutation in the prostate epithelial cells, and the oncogene activation and tumor suppressor gene inactivation is the foundation of the prostate cancer development. The formation of prostate cancer is the result of a compound biological process, which involves the transform from normal epithelial cells to cancer cells, the interaction between epithelial-stromal cells, the growth and invasion of cancer cells, and metastasis of other organs. It has been proved that the change of the microenvironment cancer and mesenchymal tissues is a common feature of cancer’s initial and progression, and the interaction between cancer and stromal cells plays an important role in the progression of cancer. The change of the mesenchyme section of cancer can destroy the homeostasis of normal prostate microenvironment, and can make an effect of the growth and invasion ability of cancer cells. The EMT (epithelial-mesenchymal transition) can contribute to decrease of apoptosis ability, and enhance of the resistant ability toward the chemotherapy, and it is one reason of the failure of curing advanced tumor. It will be profound influences for diagnosing and curing prostate cancer, that the research of studying the effect of the mesenchyme tissue on the pathogenesis and outcome of prostate cancer.Proteomics is a new subject of researching the whole proteins in organisms since 1990s, and it is a new method of high-throughput following genomics and transcriptomics. It mainly focuses on the characteristics of proteins on large-scale, including the expression level of proteins, modification after translation, the interaction between different proteins, etc. Because proteins can represent the function of genes, the research of proteomics can reveal the relationship between the change of proteins in organisms and the diseases, and provides the foundation of illustrating the pathogenesis of diseases. It has so many advanced features, that it has become a reliable method for finding biomarkers of cancers and targets of medicines, shows bright future in the diagnose and therapy of cancers.This research used the laser micro-dissection and proteomics to analysis the different expression proteins in prostate cancer and adjacent non-cancerous tissues, and differed these proteins with bioinformatics instruments using cluster analysis, to pick up the genes which were expressed in stromal tissues. For the most differentiated protein in mesenchymal tissues, MYL9, RT-qPCR and western blot analysis was used to confirm the differentiated expression between prostate cancer and non-cancerous tissues in mRNA and protein levels. After that, immunohistochemical analysis was used to amplify the expression mode of the MYL9 biomarker in large tissue microarrays. Using the pathological data and Taylor dataset, the relationship of the MYL9 gene expression and some pathological outcomes, such as pathological stage, non-recurrence biochemical survival, and overall survival, could reveal the importance of the MYL9 gene in calculating the effect on the progress and prognosis of prostate cancer. The aim of the research was to supply specific cancer biomarker, and provide some evidences about the mechanism of EMT, and help with the therapy of prostate cancer patients.Materials:1:For proteomics analysis, four fresh PCa tissues and paired 4 adjacent non-cancerous tissues of prostate were harvested from 4 PCa patients (T2N0M0 stage) who underwent radical prostatectomy and standard bilateral lymphadenectomy at Guangzhou First People Hospital from May to August 2012. For Western blot analysis, frozen samples of 20 pairs of primary PCa tissues and self-matched adjacent non-cancerous prostate tissues were collected from the tissue bank of Guangzhou First People Hospital, all patients were received radical prostatectomy between 2008 and 2013, and the age range was from 56 to 68 years old (average age was 61.31±5.65 years old). A total of 40 pairs of PCa and benign prostate tissues for RT-qPCR analysis were collected from the pathology laboratory of Massachusettes General Hospital, Harvard Medical School, patients with prostate cancer were received radical prostatectomy or TURP in 2012, and the age range was from 58 to 83 years old (average age was 67.10±6.95 years old). These patients were divided into three group,12 patients with high differentiated,15 with moderate differentiated, and 13 with low differentiated.2:Tissue microarray (TMA) including 99 primary PCa tissues and 81 adjacent non-cancerous prostate tissues was got from Shanghai Outdo Biotech Co, LTD (Cat No:HPro-Ade180PG-01), the average age was 70.71±7.99 years. Among which, the PSA level of 87 cases was higher than 4nmol/ml, and the Gleason score of 28 cases was more than 8.3. The Taylor dataset, which is a large PCa dataset with microarray expression data for mRNAs, and clinical-pathological information for the patients including 149 primary PCa tissues and 29 adjacent noncancerous prostate tissues.The study was approved by the Research Ethics Committee of Guangzhou First People Hospital. Informed consent was obtained from all patients. All specimens were handled and made anonymous according to the ethical and legal standards. None of the patients recruited in this study received chemotherapy, radiotherapy or hormone therapy before surgery.Methods:1:Four cases fresh PCa and adjacent benign tissues of prostate were obtained after the surgery immediately and frozen in liquid nitrogen and then stored at -80 C until use. Total protein was extracted from these samples by using Protein Mini Kit according to the manufacturer’s instructions. After 2D-DIGE and imaging scan analysis, the data of differential expressed proteins (fold change > 2) were obtained. The identifications of these proteins were performed by gel staining and mass spectrometric analysis.2:The ontology analysis and functional analysis of the differential expressed proteins were performed by using http://www.pantherdb.org database, http://www.string-db.org database and http://www.proteinatlas.org database. And the stroma-specific genes of prostate cancer were screened out.3:Pick out one of the most differential expressed proteins in prostate cancer stroma, then further validated its differential expressed pattern of proteomics by QPCR analysis of 40 pairs of prostate cancer and self-matched benign prostate tissues and by Western Blot analysis of 20 pairs of prostate cancer and self-matched benign prostate tissues.4:The expression pattern and localization of the selected gene in prostate cancer and non-cancerous prostate tissues were examined with immunohistochemical analysis of prostate tissue microarray samples. And the correlations of gene expression and PSA level, Gleason score, clinical-pathological stage and metastasis were investigated.5:The association of the selected gene expression with the biochemical recurrence-free survival and the overall survival of PCa patients were analyzed by using the Taylor dataset. And to further investigate whether the selected gene was involved in epithelial-mesenchymal transition of prostate cancer cells and recombination of prostate cancer stroma, the correlation of the gene and other EMT associated genes were analyzed, such as E-cadherin and Vimentin.Statistical analysis:The software of SPSS version 17.0 was used for statistical analysis. Continuous variables were expressed as x±s, the mean of two groups were compared by using paired or independent sample t test. The immunohistochemistry scores between different prostate stomal tissues were compared by Kruskal-Wallis test, and Spearman correlation analysis was also performed. Pearson’s analysis was adopted to conduct the correlation between selected gene and other EMT associated genes. For survival analysis, the first end-point was defined as biochemical recurrence and the second end-point was defined as died events. Kaplan-Meier method and log-rank test were used for analyzing the relation between gene expression and end-point events. Cox regression was applied for the univariate and multivariate analysis to determine whether the gene could be a prognostic factor for evaluating the bio-recurrence and survival of prostate cancer. Differences were considered statistically significant when P value was less than 0.05.Results:1:We found 155 differential expression proteins in prostate cancer by proteomics analysis of four prostate cancer and corresponding adjacent benign tissue, of which 66 proteins were down-regulated and 89 were up-regulated significantly. (fold changes>2). A total of 66 differentially expressed proteins, including 40 that were up-regulated and 26 that were down-regulated, were successfully identified in the PCa tissues by coomassie brilliant blue staining coupled with mass spectrometry. Among which MMC2 was the most up-regulated (Average Ratio:4.29; P=0.023) and MYL9 was the most down-regulated one (Average Ratio:-5.67; P=0.008)2:After the expression pattern searching analysis of the 66 differential expression proteins in prostate cancer by suing HPA database, we found 14 genes which mainly expressed in stroma but negative in prostate cancer cells. Among which LPP, CKB, GSTM2, GSTP1, MYL9, DPYSL3, ACTC1, GSTM3, PRPH were down-regulated and FGB, ACY1, VCL, HSPB1, MSN were up-regulated。3:Further gene function analysis of the 14 stroma genes were performed by suing http://www.pantherdb.org databases and http://www.string-db.org database showed these genes involved in different function, such as cell adhersion, cell immunity, cell metabolism, VEGF signal pathway, Wnt pathway et al. And the relationships among these genes were not obviously. Because MYL9 was the most down-regulated gene, we selected it to further study it’s clinical significance in prostate cancer.4:RT-qPCR analysis was performed to detect the expression level of MYL9 in 40 pairs of human PCa and self-matched adjacent non-cancerous prostate tissues. The expression level of MYL9 was down-regulated in PCa tissues compared with those in non-cancerous prostate tissues (PCa=1.30±0.63 vs. Benign=1.91±0.70, P <0.001, t=-4.436), and the results were consistent with the findings of proteomics.5:To validate the high-throughput data of proteomics, Western blot analysis was performed to detect the expression of MYL9 protein in 20 pairs of human PCa and self-matched adjacent non-cancerous prostate tissues. the expression level of MYL9 protein was down-regulated in PCa tissues compared with those in non-cancerous prostate tissues (PCa=0.23±0.08 vs. Benign= 0.73±0.06, P<0.001)6:Immunohistochemical analysis with large samples tissue microarray showed MYL9 was expressed mainly in the cytoplasm of stromal cells, but weakly or negatively in cancer cells or normal luminal cells of prostate tissues. The expression level of MYL9 in PCa tissues was lower than that in adjacent noncancerous prostate tissues significantly (IRS:PCa= 3.36±1.58 vs. Benign=4.85±1.02, P<0.001). The expression level of MYL9 was negative associated with the Gleason Score and clinical stage of prostate cancer (r=-0.550, P<0.001).7:The association of MYL9 expression with the biochemical recurrence-free survival and the overall survival of PCa patients were analyzed by Kaplan-Meier method, the biochemical recurrence-free survival (P=0.001) and overall survival (P =0.012) of PCa patients with low MYL9 expression were both significantly shorter than those with high MYL9 expression. The Cox hazard ratio regression model showed that there were significant differences in both the biochemical recurrence-free survival (HR=0.53,95%CI:0.30-0.74) and overall survival (HR=0.06,95%CI:0.01-0.45) rates between patients with high MYL9 expression and low MYL9 expression.8:The Pearson correlation analysis of MYL9 and E-cadherin, Vimentin, TGF-β1 and a-SMA showed MYL9 was strong correlated with E-cadherin (R=0.949, P< 0.001), and MYL9 was correlated with Vimentin (r=0.301, P=0.014), TGF-β1 (r=0.360, P<0.001) and a-SMA (r=0.336, P<0.001) in prostate cancer significantly.Conclusions:1、Proteomics can be used for discovering the differentially expressed proteins in prostate cancer, and with bioinformatics analysis we can screen out the stroma-specific genes effectively. These informations were help to investigate the mechanism of prostate cancer.2、The expression level of MYL9 in prostate cancer stroma was lower than benign adjacent cancer tissues, and the decreased expression of MYL9 in prostate cancer was involved in disease progression.3、the decreased expression of MYL9 in prostate cancer was correlated with poor prognosis of patients, it can be used as to judge the malignancy of prostate cancer.4、MYL9 was an independent factor for predicting the overall survival and biochemical recurrence-free survival of prostate cancer, and serve as an potential stromal target for therapy.5、The down-regulation of MYL9 may promote progression of prostate cancer by inducing epithelial-mesenchymal transitions of cancer cells and the recombination of prostatic stroma.