The Investigation Of Quantitative Proteomics And Platelet Derived Growth Factor-B(PDGF-B)Regulatory Network In Prostate Cancer

Purpose:To investigatethe importance of PDGF-B regulatory network in PCa progression,which will assist to understand the role and mechanisms of PDGF-B in promoting the cancer growth,provide valuable knowledge reference in the future research on anti-PDGF therapy or whether PDGF-B may a new therapeutic target of anti-angiogenesis therapies.Methods:(1)Both WT mice and TRAMP mice were sacrificed at an age of 18 weeks.The entire prostate gland was isolated from WT mice.In-solution digestion of proteins or Peptides were performed prior to the MS analysis.We performed a label free quantitative proteomics analysis combined with a careful bioinformatics analysis on the entire prostate protein extraction from TRAMP mice and compared with WT littermates.From totally 2400 identified proteins,we here presented a modest mice prostate reference proteome.containing 919 proteins.Biostatistics analysis of 515 selected proteins that were identified in all biological replicates indicated that 61 proteins presented a significant expression difference between the two groups.(2)The genotype of animals were confirmed by PCR-based assay from tail biopsies DNA.The tumorigenesis was confirmed by high field(7T)small animal magnetic resonance imaging(MRI).A sample size estimation was performed according to Lehr's formula prior to the experiment.(3)Four pairs of prostate cancer and adjacent normal prostate tissues were obtained from patients.(4)Total RNA and proteins extract from four WT mice,four TRAMP mice and human prostate tissue samples for qRT-PCR and Western Blot experiment.(5)LNCAP cells with knockdown of PDGF-B for qRT-PCR and Western blot.PDGF-BB shRNA-based knockdown in LNCAP cells grown in medium containing 10?M of PDGF-BB and Crenolanib for MTT experiment(6)LNCAP cells cultured in medium containing Crenolanib with a gradient of 10nM,lOOnM,1?M,10?M to detect cell proliferation assay.Result:The subsequent integrative bioinformatics analysis based on both qualitative and quantitative proteomics results predicted the overexpression of the platelet-derived growth factor B(PDGF-B)in tumor tissue PRDX2,PDIA3,and HNRNPL were confirmed as three novel PDGF-B-regulating proteins with immunoblot in shRNA-based PDGF-B knockdown PCa cells.The other six novel PDGF-B-regulating proteins were validated with qRT-PCR.We also proved that inhibiting PDGF-B signaling suppressed PCa cell proliferation with shRNA knockdown or induction of a PDGFR inhibitor.Urthermore,we demonstrated that Crenolanib,which is a novel PDGF receptor inhibitor,inhibited PCa cell proliferation in a dose-dependent manner.Conclusions:The overexpression of the platelet-derived growth factor B(PDGF-B)in tumor tissue and supports the hypothesis of the PDGF-B signaling network as a key upstream regulator in PCa progression.The importance of PDGF-B regulatory network in PCa progression,which will assist to understand the role and mechanisms of PDGF-B in promoting the cancer growth and provide valuable knowledge reference in the future research on anti-PDGF therapy.