The Mechanism Of Hsa＿circ＿0003258 Complexing With IGF2BP3 And Sponging Mir-653-5p To Promote The Metastasis Of Prostate Cancer

Background and purposeProstate cancer(PCa)is a commonly diagnosed cancer in men and metastasis of PCa is the main cause of death.Circular RNA(CircRNA)are single-stranded,covalently closed RNA molecules,formed by back-splicing of pre-mRNAs.Many studies have revealed that CircRNA are essential regulators of gene expression and participate in multiple biological processes of tumorigenesis via acting as miRNA sponges,RBP-binding molecules,transcriptional regulators,or templates for protein translations.However,the role of CircRNA in PCa have not been clarified.Therefore,it is urgent to explore the biological role and molecular mechanism of CircRNA in metastatic PCa.MethodsThe plasma of patients with PCa and matched normal people were sequenced to screen the differentially expressed molecule hsa＿circ＿0003258.The expression of hsa＿circ＿0003258 in plasma and tissues of PCa was evaluated using qRT-PCR and FISH.Sanger sequencing,nucleoplasmic isolation,actinomycin experiments were used to to verify the localization and circular properties of hsa＿circ＿0003258.After constructing PCa cell lines with knockdown and overexpression of hsa＿circ＿0003258,the migration ability of PCa cells in vitro was detected by transwell migration and scratch healing experiment.The metastatic ability of PCa cells in vivo was detected DU 145 cells transfected with NC or sh-has＿circ＿0003258 through lateral tail vein of BALB/c nude male mice.We constructed stable knockdown DU 145 cells using sh-hsa＿circ＿0003258 lentiviral system screened the signaling pathways that might be involved in hsa＿circ＿0003258 by using RNA-seq analysis in DU 145 cells.The enrichment of KEGG and GO pathway was analyzed.Western blot was used to verify the expression of related proteins.Subsequently,the relastionship between hsa＿circ＿0003258 and miR-653-5p was predicted online and validated their binding by double luciferase experiment,RT-qPCR,Western blot and transcriptome sequencing were used to indentified ARHGAP5 as the downstream molecule.The relastionship between hsa＿circ＿0003258 and IGF2BP3 was predicted online and confirmed by RNA pull-down,RIP and IF-FISH.HDAC4 was found to be the downstream molecule of hsa＿circ＿0003258 and IGF2BP3 by RNA-seq,Starbase and CLIP-seq.The mRNA stability of HDAC4 was explored by actinomycin experiment.the signal pathway was verified by rescue experiment.ResultsDownregulation of hsa＿circ＿0003258 significantly decreased the migratory capabilities of PCa cells and the opposite effect was observed following the overexpression of hsa＿circ＿0003258.The mice injected with cells with sh-hsa＿circ＿0003258 had less metastatic nodules in the lungs than mice injected with control cells.GO analysis showed that differential mRNAs are enriched in cell adhesion.KEGG pathway enrichment analysis indicated that the differentially expressed genes induced by hsa＿circ＿0003258 knockdown were enriched in the cell metabolism,MAPK,PI3K-AKT signaling pathway.Western blotting showed that the EMT and p-ERK levels were markedly decreased following knockdown of hsa＿circ＿0003258 and the opposite effect was observed following the overexpression of hsa＿circ＿0003258.Online database and dual-luciferase activity were performed to verify that miR-653-5p can bind to hsa＿circ＿0003258.The signal pathway was verified by rescue experiment.We confirmed the binding sites between hsa＿circ＿0003258 and IGF2BP3.Hsa＿circ＿0003258 and IGF2BP3 complex promoted EMT through stabilizing HDAC4 mRNA.Depletion of HDAC4 markedly decreased the progression of EMT and the level of p-ERK protein.ConclusionHsa＿circ＿0003258 promoted the metastasis of PCa cell.Mechanistically,hsa＿circ＿0003258 adsorbed miR-653-5p to activate ARHGAP5 expression.Moreover,we found that hsa＿circ＿0003258/IGF2BP3 enhances the stability of HDAC4 mRNA by forming RNA-protein ternary complex,thereby promoting the metastasis of PCa through ERK passway.Our findings suggest that hsa＿circ＿0003258 functions as a novel positive regulator for PCa metastasis through both hsa＿circ＿0003258/miR-653-5p/ARHGAP5 axis and hsa＿circ＿0003258/IGF2BP3/HDAC4 axis.These findings provide new insights into PCa metastasis and a novel target for PCa treatment.