Serine/Arginine Protein–Specific Kinase 2 Promotes Development And Progression By Down-regulating Numb And P53 In Pancreatic Cancer

Objective: Pancreatic cancer(PC)is one of the most lethal malignancies,that is mainly attributed to its strong local invasion,high distant metastasis rate and chemotherapy resistance.Serine/arginine protein specific kinase 2(SRPK2)phosphorylates serine/arginine(SR)domain–containing proteins and mediates the splicing activity of SR proteins,which is elevated expression in several human tumors,such as leukaemia,nonsmall cell lung carcinoma,head and neck squamous cell carcinoma,colon cancer and prostate cancer.However,the importance of SRPK2 in PC remain unexplored.Numb,a cell fate determinant,controls the function of the tumour suppressor p53.SRPK2 knockdown caused up-regulation of Numb protein in hepatocellular carcinoma(HCC),and and suppressed HCC cell proliferation,colony formation,migration and invasion.Numb prevented p53 ubiquitin degradation,and increased chemoresistance in PC cells.Thus,we investigate the expression of SRPK2 in PC tissues,and the relationship among SRPK2,Numb and p53 in PC tissues and cells.Methods: 1.In tissue level: Immunohistochemistry(IHC),western blot(WB)and Quantitative real-time Polymerase Chain Reaction(q RT-PCR)were used to detect SRPK2 expression in PC.IHC was used to investigate the relationship between SRPK2,Numb and p53 in PC tissue,and to investigate the association with clinical data and survival of PC patients.2.In cell level: Co-Immunoprecipitation(Co-IP)was used to investigate the endogenous binding of SRPK2,Numb and p53 in PC cells.Construction of stable cell lines with SRPK2 silenced and overexpressed and si RNA interference were used to detect the relationship between SRPK2,Numb and p53 in PC cells.Migration and invasion assays and CCK8 assays were used to investigate the functional relationship of SRPK2,Numb and p53 in regulating the migration,invasion and resistance to gemcitabine and oxaliplatin of PC.Results: 1.Differential expression of SRPK2 protein in PC tissues and the association of SRPK2 expression with clinical data and survival of PC patients: SRPK2 expression in 73 PC tissues was much higher than that in the paired normal pancreas,and postoperative patients with SRPK2 expression had a significantly reduced overall survival than those without SRPK2 expression as determined by Kaplan–Meier analysis,and SRPK2 expression was an independent unfavourable prognostic indicator in multivariate analysis.SRPK2 expression was positively associated with tumour T stage and Union for International Cancer Control(UICC)stage,that was not associated with age,gender,tumor location,tumor size,differentiation,lymph node metastasis,pre-therapeutic CA19-9 level and postoperative liver metastases.Spearman's rank correlation analysis showed that SRPK2 was negatively associated with Numb expression,but had no association with mutant p53(mtp53)expression in PC serial sections.2.The association of SRPK2,Numb and p53 in PC cell lines: The wild-type p53(wtp53)PC stable cell lines with SRPK2 silenced and overexpressed were successfully constructed.The SRPK2 protein level in the sg-SRPK2 groups was significantly lower than that in the corresponding scrambled RNA groups in the SRPK2-silenced stable cell lines,while SRPK2 expression in the SRPK2-GFP groups was significantly higher than that in the corresponding empty plasmid groups in the SRPK2-overexpressing stable cell lines.Without oxaliplatin treatment,silencing or overexpressing SRPK2 did not changed wtp53 protein levels.However,with the treatment of oxaliplatin,the expression of the p53 and p21 proteins was activated and significantly increased in the sg-SRPK2 group compared with the scrambled RNA group,and conversely decreased in the SRPK2-GFP group compared with the empty plasmid group. Additionally,the Numb protein level was increased in the sg-SRPK2 group vs.the corresponding scrambled RNA group,and decreased in the SRPK2-GFP group vs.the empty plasmid group,regardless of treatment with oxaliplatin.We further showed that SRPK2,Numb and p53 were co-immunoprecipitated in a triple complex in PC cells,without or with the treatment of oxaliplatin.3.SRPK2 regulated cell migration,invasion and chemosensitivity in PC cells: The migration and invasion assays showed that SRPK2 silencing decreased cell migration and invasion in the sg-SRPK2 group compared with the scrambled RNA group in PC cells,whereas SRPK2 overexpression enhanced cell migration and invasion in the SRPK2-GFP group compared with the empty plasmid group.Furthermore,the CCK8 assays showed that the silencing and overexpression of SRPK2 enhanced and decreased,respectively,the chemosensitivity of PC cells under the gemcitabine and oxaliplatin treatments.4.P53 knockdown reversed the increase of wtp53 protein,the decrease in cell migration and invasion and the increased chemosensitivity induced by silencing SRPK2 in response to oxaliplatin: Numb knockdown significantly reversed the upregulation of wtp53 protein levels induced by silencing SRPK2 during oxaliplatin treatment in PC cells.This trend was much more significant when p53 si RNA was used instead of Numb si RNA.Nevertheless,Numb or p53 knockdown did not change the SRPK2 protein level.In addition,silencing p53 significantly reversed the decrease in cell migration and invasion and the enhanced chemosensitivity induced by silencing SRPK2 in PC cells.Conclusion: Our data suggest that SRPK2 promotes the development and progression of PC,and negatively regulates wtp53 expression by downregulating Numb protein expression in response to chemical agents.SRPK2 promotes the invasion,migration,and resistance to gemcitabine and oxaliplatin of PC in a p53-dependent manner.Future research should further investigate the molecular mechanism of the interactions among SRPK2,Numb,and p53,and verify the effects of SRPK2,Numb and p53 on tumorigenesis and development through in vivo animal experiments.