The Mechanism Underlying Promoting Effect Of SMARCC1 Down-regulation On Proliferation And Metastasis In Prostate Cancer

Background and objectives:Prostate cancer(PCa)is one of most urging health problem inflicting elderly males.Most of patients with PCa are at advanced stage at initial diagnosis and androgen deprivation therapy is recommanded for those patients.Nevertheless,PCa would progress to unfavorable stage,including castration resistant and aggressive phenotype.Therefore,it is urging to determine associated molecular mechanism involved in PCa initiation and progression.SWI/SNF chromatin remodeling complex consists of multiple subunits and is also a key epigenetic factor involved in genome transcription.Mutation in SWI/SNF complex has been identified in 19%of tumor tissue and induces component loss in SWI/SNF complex to raise tumorigenesis in several cancers,including renal carcinoma and melanoma,implying important role of tumor suppressor in SWI/SNF complex.Nevertheless,there is little conclusive research about effect and mechanism of SWI/ANF complex during PCa initiaion and progression.Recent research indicates loss of SNF5 core subunit in SWI/SNF complex promotes aggressive phenotype of PCa.As for one of core subunit in SWI/SNF complex,tumor tumor suppressor role of SMARCC1 is indicated in a retrospetive research where positive staining in PCa tissue correlates with prolonged disease-free survival.In view that there is no conclusive research,we settle out research on effect of SMARCC1 on PCa.Methods:Protein profile of SMARCC1 in PCa tissue was determined by immunohistochemical assay,thereby evaluating correlation between SMARCC1 expression and parameters in clinic and pathology.Disease-free survival in PCa patients with different expression of SMARCC1 was analyzed using data from GEPIA database.Moreover,potential effect of SMARCC1 on several key events during PCa initiation and progression was also evaluated using data from GEPIA database.SMARCC1 in cell lines of PCa was knocked down using small interference sequence.Genomic transcription change was determined by RNA.Meanwhile,differential expressed genes were also filtered with standard of a P value?0.05 and a minimum of a two-fold change in expression.Then,using GO enrichment on differential expressed genes,the most affected celluar functions in PCa cell lines at transcription level were implied and further determined in phenotype and molecular assay.Phenotype assay includes experiment on proliferation and migration.In proliferation experiment,change in proliferation of PCa cell lines due to SMARCC1 loss was investigated by in vitro CCK8,flow cytometry and Edu assay as well as mouse xenograft in vivo.While migration change due to SMARCC1 loss was investigated by in vitro wound healing and transwell assay.Meanwhile,SMARCC1 induced metastasis change was evaluated by constructing mouse xenograft of lung clonization.Associated factors involved in proliferation and migration as well as activation degree of PIK3/AKT pathway was determined by western blot assay.Meanwhile,nucleus translocation of ?-catenin was determined by seperation assay on cytoplasma and nucleus.At last,PCa cell line with SMARCC1 loss was processed by inhibitors in PI3K/AKT pathway to perform function recovery experiment in the way described before to determine if the pro-oncogenesis effect of SMARCC1 loss in cell lines of PCa is mediated by over-activation of PI3K/AKT pathway.Result:No.1 Compared with PCa tissue with Gleason score?7,SMARCC1 expression level was down-regulated in PCa tissue with Gleason score>7.No.2 Disease-free survival analysis on data from GEPIA database indicated prolonged disease-free survival in PCa patients with high expression of SMARCC1.Potential inhibitory effect of SMARCC1 on several key event in PCa initiation and progression was also indicated in analysis on bioimformatic data from GEPIA database,reflecting tumor suppressor role of SMARCC1 in PCa progression.NO.3 RNA sequencing identified totally 1525 differential expressed genes with 850 up-regulated and 675 down-regulated in 22RV1,412 differential expressed genes in PC3 with 146 up-regulated and 266 down-regulated and 3220 differential expressed genes in LNCAP with 2065 up-regulated and 1155 down-regulated.GO enrichment analysis of differential expressed genes indicated SMARCC1 loss in PCa cell lines mainly affected cellular function associated with proliferation and differentiation.NO.4 SMARCC1 loss in PCa cell line induced hyper-proliferation.In vitro CCK8 and clone formation assay indicated improved proliferation in PCa cell lines with knock-down of SMARCC1.qRT-PCR and western blot assay further indicated SMARCC1 loss induced up-regulation of Cyclin as well as down-regulation of P21 and p27,members in CKI family,at protein and mRNA level.Flow cytometry and Edu assay demonstrated acceleration of cell cycle transition in PCa cell lines after SMARCC1 interference,manifested in higher percentage of cell entering G1 phase.In vivo proliferation advantage of PCa with SMARCC1 knock-down was also manifested in mouse xenograft.NO.5 Epithelial mesenchymal transition induced by SMARCC1 loss in PCa cell lines promoted migration and metastasis in PCa cell line.Immunofluorescent assay on PCa cell line with SMARCC1 loss indicated decreased fluorescet signal from epithelial marks,E-Cadherin,and increased fluorescent signal from mesechymal marks,including Vimentin and N-Cadherin.qRT-PCR and western blot assay indicated same change in epithelial and mesenchymal marks as well as up-regulation in epithelial mesenchymal associated transcription factors,including snail,slug and zeb1.Meanwhile,wound healing and transwell assay in vitro indicated improvement in migration ability in PCa cell line with SMARCC1 loss.In vivo mouse xenograft of lung clonization demonstrated metastasis advantage of PCa cell lines with SMARCC1 loss.NO.6 Elevated phosphorylation level in site thr 308 and ser 473 indicated over-activation of PI3K/AKT pathway in PCa cell lines with SMARCC1 loss.Meanwhile,we also detected the nucleus transcription of Beta catenin,which can regulate proliferation and epithelial mesenchymal transition in PCa.Blockade to PI3K/AKT pathway with LY294002 can reverse pro-oncogenesis effect of SMARCC1 loss on PCa,implying over-activation of PI3K/AKT pathway mediates pro-oncogenesis effect due to SMARCC1 loss in PCa.Conclusion:SMARCC1 is one of tumor suppressor during PCa progression.SMARCC1 loss in PCa induces hyper-proliferation and epithelial mesenchymal transition in PCa mediated by over-activation of PI3K/AKT pathway.