RNA Binding Protein Musashi2 Stabilizing Androgen Receptor Drives Prostate Cancer Cell Proliferation

Objective: Androgen deprivation therapy(ADT)that blocks the androgen receptor(AR)pathway is a basic treatment for advanced prostate cancer.However,after 18-24 months patients progress to castration-resistant prostate cancer(CRPC),which leads to treatment failure.Therefore,there is an urgent need to find key molecules that interact with AR as a new strategy for treating prostate cancer and even CRPC.Methods: 1.The data in the GEO databas was used to compare the expression differences of multiple RNA-binding proteins(RBPs)between prostate cancer and normal prostate tissues by bioinformatics technologies.The RBPs with significant differential expression were screened and then the correlation between RBPs and AR expression were analyzed.As a result,Musashi2(MSI2)was selected to detect its correlation with AR and AR downstream molecules NKX3.1,KLK3 and TMPRSS2 through bioinformatics analysis.Furthermore,immunohistochemistry(IHC),immunofluorescence(IF),western blotting were used to detect the expression of MSI2 and AR in prostate cancer tissue,tissue chips and prostate epithelial cell lines,the relationship between MSI2 and Gleason scores(GS)as well as the relationship between AR and MSI2 expression level.Establish the MSI2 knockdown stable cell lines using LNCa P/C4-2B/22RV1 cell lines.And then qRT-PCR and western blotting were used to detect the mRNA and protein levels of AR and AR downstream molecules.2.Colony forming experiments and CCK-8 experiments were used to detect the proliferation function differences of LNCa P/C4-2B/22RV1-MSI2 knockdown stable strains in vitro.MSI2 knockdown stable strains were transfected with AR overexpression plasmid and CCK-8 experiments were used to detect the proliferation rate.We performed transwell experiments to detect the migration and invasion ability of stable strains.Tumor xenografts experiments of MSI2 knockdown stable strains were used to determine the tumorigenicity function,tumor volume and weight.Tumor tissues were collected,and western blotting was applied to detect MSI2 and AR expression level.3.RNA Decay experiments were performed to detect the regulation of MSI2 on AR mRNA stability;RIP experiments were used to detect the direct binding relationship between MSI2 and AR mRNA.According to reported MSI2 binding sequence,the possible binding sites of AR mRNA UTR sequences were selected to construct the binding sites carrying reporter vector plasmids.And then perform luciferase reporter gene experiments to determine the binding site.Furtherly,mutate the binding sites and construct reporter gene plasmids carrying the mutant binding sites,and then perform luciferase reporter gene experiments to further determine the binding site.Finally RIP experiment,RNA pulldown experiments verified the MSI2 binding site in AR UTR sequence.CHX experiments were applied to detect the regulation of MSI2 on AR protein stability.The direct binding relationship between MSI2 and AR was detected by Co-IP.Furtherly,MG132,3-MA or chloroquine was added separately to detect AR expression in MSI2 knockdown strains.Results: 1.Using the bioinformatics technology,twelve significantly differently expressed RBPs were selected between prostate cancer and normal prostate tissues.Further study was performed to determine the correlation between RBPs and AR expression,and found that MSI2 was highly correlated with the expression of AR and AR downstream molecules NKX3.1,KLK3 and TMPRSS2.IF,IHC,and western blotting were used to determine that the AR expression levels were highly correlated with that of MSI2.MSI2 was upregulated in prostate cancer tissue and significantly correlated with GS.qRT-PCR and western blotting tests revealed that mRNA and protein levels of AR were reduced in MSI2 knockdown strains.2.Downregulation of MSI2 in both androgen sensitive LNCa P cell line and androgen insensitive C4-2B/22RV1 cell lines inhibited tumor formation in vivo and decreased cell growth in vitro,which could be reversed by AR overexpression.Transwell experiments showed a reduced migration and invasion ability of MSI2 knockdown strains.3.RNA Decay test showed that MSI2 positively regulated the stability of AR mRNA,and RIP test verified that MSI2 directly binded to AR mRNA.Based on the reported MSI2 binding sequence,select the twenty possible binding sites of AR mRNA UTR sequence and construct reporter vector plasmids carrying binding sites.The luciferase reporter gene experiment narrowed the twenty binding sites down to three.Then luciferase report experiments using reporter gene plasmids carrying the mutant binding sites determined the binding site as M15.Finally,RIP experiments and RNA pulldown experiments verified the direct binding relationship between M15 and MSI2.CHX concentration gradient experiments detected that the protein stability of AR and downstream molecules PSA and NKX3.1 significantly decreased after MSI2 knockdown.Co-IP detected that there was no direct binding relationship between MSI2 and AR.Only MG132 could reverse the reduction of AR expression in MSI2 knockdown strains rather than 3-MA or chloroquine.It is speculated that MSI2 can upregulate AR protein stability by proteasome-mediated AR degradation.Conclusion:1.MSI2 is highly expressed in prostate cancer,especially in tumor tissues with high GS.AR expression levels are highly correlated with MSI2.2.The down-regulation of MSI2 in androgen-sensitive and insensitive prostate cancer cells can inhibit tumor formation in vivo and downregulate cell growth in vitro.And AR overexpression can reverse the above process.3.Mechanistically,MSI2 directly binds to the 3'-UTR of AR mRNA,enhancing its stability and thus upregulating its transcriptional activity.MSI2 may strengthen AR protein stability by inhibiting proteasome-mediated AR degradation.