Effect Of NPRL2 On The Sensitivity Of Olaparib In Castration-resistant Prostate Cancer

Objective: To explore the effect of NPRL2 on improving the sensitivity of Olaparib in castration-resistant prostate cancerMethods: Western Blot assay for NPRL2 protein in normal prostate epithelial cells(RWPE-1),prostate cancer cell line(LNCaP),prostate cancer cell line(PC3),and enzaliline-resistant castrated prostate cancer cells(LNPER)Express the situation.Overexpression and interference of NPRL2 were established by transfection of lentivirus into CRPC cells.The overexpression and interference efficiency of NPRL2 were verified by real-time fluorescent PCR and Western blot.Cell proliferation and cytotoxicity were detected by CCK-8 method,apoptosis was detected by flow cytometry,and cell invasion and metastasis were detected by Transwell invasion and migration chamber assay.In addition,we investigated NPRL2 overexpression and silencing affecting apoptosis-related proteins(cleaved caspase-3,BAX and Bcl-2),ataxia telangiectasia-mutated(ATM)proteins and epithelium Expression of Epithelial-mesenchymal transition(EMT)-associated proteins(E-cadherinprotein and vimentin protein).In addition,we performed in vivo experiments to verify the results of changes in NPRL2 related proteins in regulating Olaparib sensitivity.Results: NPRL2 regulates the sensitivity of castrated resistant prostate cancer(CRPC)to Olaparib;Western blot shows that NPRL2 is underexpressed in RWPE-1 cells and is highly expressed in LNCaP,PC3 and LNPER cells and successfully constructed a stable strain that overexpresses and interferes with NPRL2 QPCR and Western blot results showed that lentiviral transfection can significantly increase and decrease the mRNA and protein levels of NPRL2.CCK8 experiments showed that NPRL2 overexpression promoted the growth of CRPC cells and resistance to Olaparib,and NPRL2 silencing inhibited the proliferation of CRPC cells and enhanced the sensitivity to Olaparib.Flow cytometry and Transwell experiments showed that silencing NPRL2 combined with Olaparib significantly promoted apoptosis and prevented cell invasion and metastasis.Western blot results showed that the expression of cleaved caspase-3,BAX protein and E-cadherin protein and the expression of Bcl-2 protein and vimentin protein were decreased after silencing NPRL2 combined with Olaparib.Silencing NPRL2 down-regulated p-ATM and ATM protein expression in CRPC cells,and up-regulated p-ATM and ATM protein expression levels.In vivo experiments demonstrated that Olaparib significantly inhibited the subcutaneous tumor growth of PC3 cells in nude mice after silencing NPRL2.Western blot analysis showed that subcutaneous tumors increased apoptosis and DNA damage increased after silencing NPRL2.Conclusion: Silencing NPRL2 enhances the sensitivity of CRPC to Olaparib,and NPRL2 can serve as a potential therapeutic target and predict CRPC resistance to Olaparib.