NPRL2 Influences The Sensitivity Of Castration-resistant Prostate Cancer To Niraparib And Its Mechanism

PART I: BIOINFORMATICS ANALYSIS OF NPRL2 EXPRESSION AND PROGNOSTIC VALUE IN PROSTATE CANCERObjective: To perform bioinformatics analysis of the NPRL2 gene in prostate cancer using public databases.Methods: RNA-SEQ data from TCGA-PRAD and GTEx-normal prostate and their clinical data were retrieved using the UCSC Xena browser to analyze the expression of NRPL2 in prostate cancer and its impact on patient prognosis,to plot the ROC curve of NPRL2 in prostate cancer,and to perform immunohistochemistry in prostate cancer tissue microarrays.Results: By comparing the transcriptional profiles of NPRL2 gene in TCGA-PRAD and GTEx-normal prostate tissues,we found that the transcriptional product ENST00000232501.7 of the protein encoded by NPRL2 gene only accounted for about 20% of the total transcripts in normal prostate tissues,however,in prostate cancer tissues and paracancerous tissues,this proportion increased dramatically to more than60%.In addition,we confirmed that the absolute expression of this transcript was significantly higher in prostate cancer(log2(TPM+0.001)).The median expression value of NPRL2 was taken as the cut-off point.DSS and OS were found to be significantly shorter in the NPRL2 high expression group than in the NPRL2 low expression group(The P values were 0.034 and 0.021).there was a shortening of PFS in the NPRL2 high expression group,but the difference was not statistically significant.We then plotted the ROC curves of NPRL2 in prostate cancer by TCGA,and the variable NPRL2 had some accuracy in predicting Normal and Tumor outcomes(AUC = 0.759,CI=0.695-0.822).Finally,we then performed immunohistochemical staining in prostate cancer tissue microarrays to verify NPRL2 expression at the protein level.Similar results were also found for high expression of NPRL2 in prostate cancer.Conclusion: The typical protein-encoding isoform of NPRL2(ENST00000232501.7)is significantly upregulated in prostate cancer tissues and is associated with poor survival in patients with prostate cancer,and NPRL2 may play an important biological role in the development and progression of prostate cancer.PART II: STUDY OF NPRL2 AFFECTING SENSITIVITY TO NIRAPARIB IN CASTRATION-RESISTANT PROSTATE CANCERObjective: To investigate whether NPRL2 can influence the sensitivity of castration-resistant prostate cancer(CRPC)to Niraparib.Methods: GSEA was performed in primary PRAD cases in TCGA by setting median NPRL2 expression as the cutoff.CRPC cell lines stably silencing NPRL2 were constructed by lentiviral transfection,and the knockdown efficiency was verified by q RT-PCR and Western blot.To observe the effect of NPRL2 on the survival of CRPC cells after Niraparib treatment,CCK-8 assay was then conducted to explore dose-dependent niraparib sensitivity.Further apoptosis was detected by flow cytometry.In addition,the expression changes of apoptosis-related proteins(Cleaved caspase-3 protein,Bcl-2 protein and Bax protein)in NPRL2-silenced CRPC cells after Niraparib treatment were detected by Western blot.Results: GSEA of NPRL2 expression revealed that the high NPRL2 expression group was significantly enriched in the two gene sets "DNA repair" and "oxidative phosphorylation"(P<0.05).The results of q PCR and Western blot showed the successful construction of human prostate cancer PC3 and DU145 cell lines with stable NPRL2 silencing,and the results of CCK-8 colorimetric assay showed that the survival rate of PC3 and DU145 cells with NPRL2 silencing was significantly lower under Niraparib treatment compared to PC3 and DU145 cells without NPRL2 silencing.survival rate was significantly reduced under Niraparib treatment.The results of flow cytometry showed that silencing NPRL2 increased Niraparib-induced apoptosis in PC3 and DU145 cells after treatment with Niraparib(100 n M)or DMSO.Western blot results showed that PC3 and DU145 cells silenced with NPRL2 by After Niraparib treatment,the expression levels of cysteinase-3(Cleaved caspase-3)and Bax(only in PC3,DU145 lacked Bax)were higher than those of cells without NPRL2 silencing,while the expression levels of Bcl2 were lower than those of cells without NPRL2 silencing.Conclusion: NPRL2 affects the sensitivity of CRPC cells to Niraparib.PART III:THE MECHANISM OF NPRL2 AFFECTING THE SENSITIVITY OF CASTRATION-RESISTANT PROSTATE CANCER TO NIRAPARIBObjective: To explore the mechanism of NPRL2 affecting the sensitivity of castration-resistant prostate cancer to Niraparib.Methods: We constructed a gene-gene function interaction network centered on NPRL2 using the Gene MANIA(http://genemania.org/)website.It was observed that NPRL2 may have a functional relationship with UBE2 M.Subsequently,bioinformatic blood analysis,cellular immunofluorescence assay,immunohistochemical examination,Co-Immunoprecipitation,MG132 and Cycloheximide(CHX)assay,ubiquitination assay,and nude mouse animal model were used to explore the possible mechanisms by which NPRL2 affects the sensitivity of CRPC cells to Niraparib.Results: 1.NPRL2-centered gene-gene functional interaction network was observed and NPRL2 was found to have a possible functional relationship with UBE2 M.Immunofluorescence examination observed co-localization of both proteins,NPRL2 and UBE2 M,in PC3 and DU145 cells.In addition,immunohistochemical staining on prostate cancer tissue microarrays revealed a similar location of distribution in NPRL2 and UBE2 M cells.Further,immunoprecipitation experiments were used to confirm that NPRL2 interacted with UBE2 M in PC3 and DU145 cells.By MG132 and Cycloheximide(CHX)assay and ubiquitination assay,we speculated that UBE2 M may stabilize NPRL2 by reducing polyubiquitination and proteasomal degradation of NPRL2,and it may be because UBE2 M makes the relatively stable expression of NRPL2 and thus affects the sensitivity of CRPC cells to Niraparib.2.The results of CCK8 method,flow cytometry and Western blot results can be concluded that UBE2 M also affects the sensitivity of CRPC cells to Niraparib.Silencing of NPRL2 or UBE2 M significantly enhanced the inhibitory effect of Niraparib on the growth of tumor volume,affected the proliferation of tumor cells and induced apoptosis in nude mice transplantation tumor animal models.3.Western blot and flow cytometry experiments showed that NPRL2 enhanced Neddylation through UBE2 M and thus affected the sensitivity of CRPC cells to Niraparib.Conclusion:This study identified a novel NPRL2-UBE2 M complex that modulates the sensitivity of CRPC cells to Niraparib.Therefore,targeting NPRL2 and blocking the NPRL2 pathway could be an adjuvant strategy for PARPI therapy.