Enhanced Expression Of SRPK2 Contributes To Aggressive Progression And Metastasis In Prostate Cancer

Background and Objective:Prostate cancer(PCa)has become one of the most diagnosed non-cutaneous tumor.The incidence and mortality of PCa in developed countries,especially American,is continuously increasing during last two decades.An upward trend in incidence rates and mortality rates was also observed in China PCa patients.PCa is indeed a clinically heterogeneous and multifocal cancer which need to be addressed,however,a minority of PCa patients will get biochemical recurrence(BCR)and eventually develop castration resistant PCa(CRPC)or metastasis which is a fatal progression to patients.Therefore,it is important to identify more molecular mechanism for better understanding the progression and metastasis,and provide valuable knowledge for preventing from progression and improving diagnosis of PCa.Serine/Arginine-rich(SR)proteins are pre-mRNA processing proteins that can regulate the alternative splicing and have been reported to be associated with splicing dysregulation in various cancers.The prototypical SR protein,for example,of alternative splicing factor/splicing factor 2(ASF/SF2)has been found to be oncogenic and affect on cellular transformation in several malignancies such as lung cancer,breast cancer and PCa.Furthermore,SR Protein-Specific Kinase(SRPKs),such as SRPK1 and SRPK2,could phosphorylate ASF/SF and involve in the regulation of cell cycle.Previous study find that inhibition of SRPK1 and subsequent negative effect in tumour angiogenesis by regulating VEGF isoform could suppress PCa growth.As for SRPK2,in addition to its major role in cell cycle regulation and cell apoptosis,it could also promote the cancer cells growth and migration.While resisting cell death,tumor invasion and tumor metastasis,sustained angiogenesis are major hallmarks of cancer,the functions above indicated SRPK2 might play an important role in tumor progression and metastasis.Up-regulation of SRPK2 has also been found in leukemia,colon cancer,lung cancer,head and neck squamous cell carcinoma and hepatocellular carcinoma,however,there are no previous reports on its involvement in PCa.We therefore aimed to investigate the role of SRPK2 in PCa progression and metastasis.Materials and Methods:1.Cell lines transfections and clinical tissue samples Three PCa cell lines(LNCaP,DU 145 and PC3)were all purchased from ATCC,cultured according to normal standard.The stable cell line of over-expression SRPK2 and control were made by HYY Med Company based on antivirals with the ORF sequence of SRPK2.For IHC analysis,a tissue microarray(TMA)of 80 cases was purchased from Creative Bio array(USA.Catalog number:PRTMA053).For further evaluation and survival analysis of SRPK2,a publicly available dataset named The Cancer Genome Atlas(TCGA)(GEO:GSE21032)with mRNA microarray expression data and relevant clinical information was collected.2.Immunohistochemistry was for SRPK2 expression evaluation in TMA.A final nonreactive score(IRS)was obtained by adding percentage and intensity of SRPK2 protein staining.IHC was also used to preliminarily identify the role of SRPK2 and vegf-a vascular regulation.3.Expression levels of SRPK2 protein in cells lines with affections of SRPK2-overexpression plasma and corresponding control were detected by Western blot analysis and QPCR.4.Effects of SRPK2 in cell proliferation,migration and invasion of PCa cells were detected by CCK-8 assay,wound-healing assay and transwell assay.5.The effect of SRPK2 on cell cycle and cell apoptosis were determined and analyzed by cell cycle assay and cell apoptosis assay Kit.6.Generation of the in vivo xenograft model was used to investigate the effect of SRPK2 on tumorigenesis and growth in vivo.7.Bioinformatics softwares were used to preliminarily discuss the potential functions of SRPK2 and the next possible direction.Statistical analysis:The SPSS version 19.0 software for Windows(SPSS Inc.,Chicago,IL,USA)was used for all statistical analysis in the current study.All the continuous variables and data were expressed as the means 土 SD.Associations between SRPK2 expression and various clinicopathological characteristics were evaluated by two independent samples t-test and One-way ANOVA test.Kaplan-Meier and Cox proportional hazards regression analysis was then used for survival analysis.Differences were considered statistically significant when the P-value was less than 0.05.Results:1.Expression of SRPK2 protein is increased in human PCa tissues.To assess the SRPK2 expression profiles in PCa tissues,we evaluated some public microarray datasets comprising different PCa samples by using Oncomine cancer database.The results showed that SRPK2 expression in human PCa tissues was evidently higher than that in normal prostate tissues.In addition,a TMA including prostate cancer and prostate tissues samples was immunohistochemically stained for SRPK2 expression evaluation.Expression level of SRPK2 in PCa tissues was evidently higher when compared to adjacent benign prostate tissues(P=0.023).2.SRPK2 expression associates with advanced progression,metastasis and unfavorable prognosis in human PCa.In this study,It was interesting to find that SRPK2 protein were significantly progressively increased with increasing tumor grades,specifically when Gleason score≥8.High expression of SRPK2 staining was also significantly associated with the higher advanced pathological stage.Similar to the results from TCGA dataset,increased SRPK2 expression was associated with higher Gleason Score,advanced pathological stage,presence of metastatic tumor,which showed the positive correlation between the high expression of SRPK2 and aggressive progression and metastasis.Using TCGA public dataset,Kaplan-Meier survival curves were constructed to stratify patients survival.A significant difference in the survival curves of SRPK2 high and low expression in PCa patients was identified for biochemical recurrence-free survival(P=0.016),however there was no significant difference for the overall survival.Subsequent univariate and multivariate Cox proportional hazards models identified no significant associations between increased SRPK2 expression and biochemical recurrence-free survival.3.SRPK2 promotes cell proliferation,invasion,migration and cell cycle,but inhibit apoptosis of PCa in vitro.4.SRPK2 promotes tumor growth of PCa in vivo xenograft mode.And tumor tissue suggests that SRPK2 has a potential effect in promoting regulation of vegf-a.Conclusions:1.SRPK2 can discriminate patients with prostate cancer from patients with benign prostate hyperplasia,suggesting that it may serve as a tumor promoter.It also can predict the probability of early biochemical recurrence,which may help to choose the better and effective individualized treatment for patients.2.The enhanced expression of SRPK2 may correlate with clinical progression and may be a prognostic factor of aggressive progression and metastasis of PCa3.SRPK2 may effect the progression and prognosis of prostate cancer through regulating the cell proliferation,migration,invasion,cell cycle and apoptosis of tumor.At the sae time,it may also affect the tumour angiogenesis.4.SRPK2 may also promote the regulation of tumor angiogenesis and further promote cancer progression and metastasis.