| Background and objectives:As one of the most common malignant tumor in western countries, prostate cancer caused the second death rate in male cancers in the world, more than 900 thousand people were newly diagnosed prostate cancer patients. By 2015, the number of new cases of prostate cancer in the United States reached 220 800, accounting for 26% of the number of male malignant tumor cases, the highest first, death cases is 27 540 cases, accounted for 9% of malignant tumor caused male deaths, the second largest cause of death among the male malignant tumor. Compared to the developed countries in Europe and America,the incidence of prostate cancer in china is lower, but in recent years, with the change of diet structure,improved living conditions and the population aging, the prostate cancer incidence and mortality showed a clear upward trend, In 1997-1999, prostate cancer incidence of Shanghai has elevated about 3.5 times higher than which in 1985-1987. With the development of diagnostic and therapeutic technologies, most early-stage prostate cancer patients underwent radical surgery or castration therapy and extended their life span, but 20% -50% of these patients had biochemical recurrence (BCR Biochemical recurrence) after surgery, While the effects of remedial treatment (radiotherapy, chemotherapy) are not ideal. In today’s prostate cancer detection, PSA, Gleason, TMN stage, hormone receptor,positive margin and other indicators have been used to predict the biological behavior of prostate cancer.these indicators help people foresee the prognosis of prostate cancer, but these indicators,either alone or in combination, can not accurately diagnose recurrence and metastasis, Therefore, it is urgent to find a more specific, more effective clinical or pathological marker in the field of tumor molecular biology to predict the prognosis of prostate cancer.Cytokinesis regulatory protein 1 (Protein regulator of cytokinesis 1 PRC1) is one of the 31 CCP gene, which is mainly expressd in the G2/M phase.when cells enter the Gl phase or exit mitosis, PRC1 protein expression was significantly decreased. Previous studies showed that PRC1 directly negative controlled by the tumor suppressor gene P53. PRC1 are also usually treated as a malignant proliferation marker and therapeutic target in the study of breast cancer, bladder cancer, cervical cancer and other tumors, but the relationship between PRC1 and prostate cancer prognosis is still unclear.In our study, we first studied the role of PRC1 in tumor progression through review of the literature, followed by QPCR and Western blot proved PRC1 upregulated in mRNA and protein level in prostate cancer cell lines.We analyzed the differential expression of PRC1 between cancer tissue and non-cancerous tissues in prostate cancer tissue microarray with immunohistochemical. Subsequently,we analyzed PRC1 mRNA expression in cancer tissues and non-cancerous tissue in microarray database-Taylor database (NCBI GEO accession:GSE21032). a comprehensive analysis about the correlation between PRC1 and pathological features of prostate cancer was made at the same time. In the end, PRC1 joint of the current clinical indicators such as PSA, Gleason score were utilized to build a model which could predict the biochemical recurrencer of Pea, Statistically illustrated that PRC1 can be used to predict prognosis of prostate cancer。Materials and Methods:Cell culture:prostate cell line PC3, LNCaP, BPH was purchased from ATCC.Cells was cultured with medium containing 10% fetal calf serum and 1% of the double anti, culture box adjusted to 37℃,5% carbon dioxide.Quantitative real-time polymerase chain reaction (RT-qPCR):Total RNA was extracted from hyperplasia of prostate cells and prostate cancer cells according to the kit specification and 2ug total RNA was reverse transcribed, real-time fluorescence quantitative PCR uesd 20ul system (PRV); 2000 system (Corbett research, Mortlake, Australia). Data analysis completed by rotor gene V5.0 (Corbett research), Instrument attached experiment scheme contains the concrete operating methods.Western blot:Extracting protein from hyperplasia of prostate cells and prostate cancer cells,2 ug protein was taken in SDSPAGE gel for electrophoresis then transferred to the hybrid nitrocellulose membrane, with 5% skimmed milk powder soluble in TBST blockd for 1 hour, then washing membrane.diluting PRC1 antibodies (rabbit monoclonal antibody, ab51248, Abcam Co Ltd., USA) and beta actin antibodies (sc-47778, Santa Cruz, USA) according to the scale of 1:2000.after all night long incubation, observation was completed with SuperSignal West PICO chemiluminescence detection system, and beta actin was choosed as internal protein.Immunohistochemical analysis:Clinical samples are stored in 10% neutral formalin for paraffin embedding, the tissues are cutted into thickness 4um slices and then xylene remove paraffin, hydration (Dako envision system (Dako diagnostics, Switzerland). Protease digestion and blocking endogenous peroxidase, according to the proportion of 1:200 diluted antibody (rabbit monoclonal antibody, ab51248, Abcam Co Ltd., USA) and incubating antibody in 4℃ fridge overnight, wash slice at the second day, finging the target protein staining using horseradish peroxidase labeled polymer.the blank control without primary antibody treatment, the immune staining were scored by two pathologists independently on the organization after hematoxylin counterstain and the two doctors don’t know the diagnosis and prognosis of patients. Then compared the two groups of scoring data, any difference in the score of the specimens should be re evaluated, the immune staining score was completed until the score of two physicians reached a consensus,. Scoring methods are as follows:(taking ten typical field of vision in the microscope and counting the number of positive staining cells, calculate the proportion of positive cells, according to the antibody specification, cytoplasmic staining is positive. Assess the level of protein expression according to the proportion of stained cells (0-100%) and the depth of staining (0-negative,1-positive,2-positive,3-positive) in each tumor tissue section. The proportion of stained cells and the depth of staining were added together to form the final immunohistochemical score, IRS.Statistical analysis:All experimental data were expressed as mean ± standard deviation, statistical analysis of data processing with SPSS 17.0 statistical software package (SPSS Inc, IL, USA). Independent samples T-test results indicated with all 2 x 2 table by Fisher’s exact test for 2 x 2 were non-Pearson chi-square test, using the Kaplan-Meier method and the Log-rank test was used for survival analysis, Cox regression model for risk factors univariate analysis and multivariate survival analysis, when P value less than 0.05 was considered a statistically significant difference.Results:1.we selected PRC1 from the CCP gene cluster.We analyzed the 31 CCP genes in the Taylor database with Kaplan-Meier plots, the results showed 10 up-regulated genes is associated with biochemical recurrence. In the 10 genes. Previous studies showed that PRC1 may be an predictor of malignant tumor cells proliferation, while at the same time, previous studies have also reported the gene is associated with poor prognosis in a variety of human cancers.2. PRC1 is up-regulated in prostate cancer cell lines.The experimental results of QPCR and Westernblot showed that the expression of PRC1 in prostate cancer cells was significantly higher than that in the mRNA and protein levels in benign prostate cells (BPH-1).3 The expression of PRC1 protein in clinical specimens of prostate cancer increased. Immunohistochemistry results showed that PRC1 protein was expressed mainly in the cytoplasm of the cancer tissue, and did not express or just expressed moderate in adjacent noncancerous tissues. The PRC1 protein expressed in prostate cancer tissues is significantly higher than that of non cancer tissues with immunohistochemical score (IRS):IRS:PCa=5.56+1.624vs.Benign=4.65 plus or minus 135.2, P< 0.001).4 PRC1 mRNA expression is correlated with the clinicopathological features of prostate cancerOur clinical tissue chip immunohistochemistry confirmed the up-regulated expression of PRC1 proteins, and in gene chip Taylor, PRC1 mRNA expression in prostate cancer tissue was also significantly higher than those in non cancer tissues (PCa=6.8734+0.3802 vs. Benign=6.6129+0.1239, P&It; 0.001). More significant, the increased expression of PRC1 is associated with high Gleason score (P= 0.041) and tumor metastasis appeared (P= 0.006), PRC1 is also closely related to biochemical recurrence (P=0.013). All these information indicates that PRC1 and the clinical prognosis of prostate cancer are closely linked.5 The analysis of mRNA expression of PRC1 is helpful to predict the prognosis of prostate cancerWe used Kaplan-Meier to analyze the association between the PRC1 gene expression and the overall survival rate and biochemical recurrence rate in patients with prostate cancer in the Taylor database, the median of PRC1 expression was choosed as the point of division and prostate tissues could be divided into high expression group (75 cases) and low expression group (75 cases). For the medium-term forecast of prostate cancer survival rates, there was no statistical difference between the PRC1 high expression group and low expression group 。 But in the study of biochemical recurrence, the high expression group had a significantly earlier biochemical relapse ration. In COX regression analysis, Analysis of high expression group and low expression group on biochemical recurrence time also showed significant differences in univariate (HR 5.08,95% CI 2.51-10.26; P＜0.001). In addition, in the multivariate analysis, the upregulation of PRC1 (HR 6.23,95% CI 2.08-19.06; P= 0.001), high Gleason score (HR 2.70,95% CI 1.69-4.33; P< 0.001), preoperative PSA level (HR 1.01,95% CI 1.00-1.01; P= 0.02) all are independent predictors to biochemical recurrence.Conclusion:1.PRC1 is up-regulated in prostate cancer cells and tissues, according to the difference in PRC1 expression of mRNA and protein level, the identification of prostate cancer and benign prostatic tissue could be easily completed.2 Increased PRC1 expression is associated with the pathological characteristics of prostate cancer, Clinical quantitative detection to PRC1 can help predict the prognosis of prostate cancer.3. Prostate cancer patients’PRC1 expression is closely related to the early biochemical recurrence, The evaluation of PRC1 expression contributes to the prediction of biochemical recurrence of prostate cancer.It could provide effective information for clinicians to formulate individualized treatment plans. |
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