Mechanism Of M6A Modification Mediating CircRBM33-FMR1 Complex Formation To Enhance Mitochondrial Respiration For Prostate Cancer Progression

BackgroundThe M6A modification is the most abundant RNA modification on mammalian mRNA and has been shown to be aberrantly modified in numerous tumors,including prostate cancer.Recent studies have found that a variety of non-coding RNAs,including circular RNAs,also have abundant m6A modification sites.Although abnormally expressed circRNAs have been reported to play a role in promoting the progression of prostate cancer,it remains unclear whether m6A modification is involved in the abnormal regulation of circRNAs on prostate cancer.Therefore,the recent study mainly explores the regulatory role of m6A-modified circRBM33 in the progression of prostate cancer,and further elucidates the specific molecular mechanism of m6A modification in it.MethodsTo screen out circRNAs associated with prostate cancer biochemical recurrence via setting the expression abundance threshold(PFKM>0.5)and applying the logistic regression analysis(log Rank P<0.05)from the circRNA sequencing data of prostate cancer downloading from GEO databases.Next,combine the above data with our m6A-meRIP sequencing for Venn intersection analysis to identify the target circRNA for the current study.Sanger sequencing was applied to identify the circularization site of circRBM33.MeRIP-qPCR to examine the enrichment of circRBM33 in m6A-modified antibodies.KM plot analysis to explore the relationship between circRBM33 expression and biochemical recurrence of prostate cancer.The source of circRBM33 was verified by convergent and divergent primers;RNase R and actinomycin D treatments were used to investigate the stability of circRBM33.The stable circRBM33 overexpression and low expression cell lines were constructed by lentivirus.The gain or loss of function of circRBM33 was detected in vivo and in vitro by CCK-8,plate clone,Transwell assay and mouse subcutaneous xenograft tumor assay.FISH and nuclear cytoplasmic separation assays were used to detect the cellular sub-localization of circRBM33.FISH combined with immunofluorescence,ChIRP and RIP experiments verified the co-localization and interaction between circRBM33 and FMR1.METTL3 specific inhibitor-STM2457 or loss of METTL3 function were used to investigate the effect of m6A on the tumor-promoting effect of circRBM33 and the interaction mechanism between circRBM33 and FMR1.IHC and FISH verified the clinical relevance of circRBM33 and FMR1.FMR1-RIP sequencing,KEGG analysis,qPCR and WB identified the downstream pathways and target genes regulated by circRBM33-FMR1 complex.Rescue experiments were used to analyze the effects of the circRBM33-FMR1/PDHA1 regulatory axis on the acetyl-CoA level,ATP production,NAD+/NADH ratio,oxygen consumption rate,and biological functions of prostate cancer cell lines.The enrichment of PDHA1 was detected by FMRI-RIP experiment.The effect of circRBM33 or FMR1 on PDHA1mRNA stability was detected by actinomycin D assay.ResultsThe only intersection of Venn intersection analysis was circRBM33,the target molecule of this study.The back-splicing site of circRBM33 is composed of exons 2 and 5.CircRBM33 can be significantly enriched by m6A-modified antibodies and is associated with poor prognosis of prostate cancer.CircRBM33 is derived from cDNA rather than genomic DNA.CircRBM33 was more resistant to RNase R digestion than the linear product of its parent gene RBM33 transcript and was more stable under actinomycin D treatment.The stable circRBM33 overexpression and low expression cell lines were successfully constructed and the expression vector had little effect on the expression of the parent gene RBM33.In vivo and in vitro functional experiments showed that circRBM33 promoted the proliferation and metastasis of prostate cancer,and could be recued by STM2457.FISH and nuclear plasma separation experiments showed that circRBM33 was mainly localized in the cytoplasm.FISH combined with IF experiments showed that circRBM33 and FMR1 had a co-localization relationship.ChIRP and RIP experiments confirmed that circRBM33 interacted with FMR1 through the m6A modification site and knocking down the expression of METTL3 could weaken this interaction.IHC and FISH analysis showed that the expressions of circRBM33 and FMR1 were positively correlated and both were positively correlated with Gleason score.Overexpression of circRBM33 can increase acetyl-CoA levels,ATP production,reduce NAD+/NADH ratio,and increase oxygen consumption rate in prostate cancer,and its phenotypic function can be rescued by FMR1 or PDHA1.FMR1-RIP experiments showed that PDHA1 could be significantly enriched.The CircRBM33-FMR1 complex increased PDHA1 mRNA stability.ConclusionCircRBM33 is highly expressed in prostate cancer cell lines and tissues and correlates with high Gleason scores.Prostate cancer patients with high expression of circRBM33 were associated with shorter time to biochemical recurrence.CircRBM33 interacts with FMR1 to form a binary complex through the m6A modification site,and jointly regulates the stability of PDHA1 mRNA and upregulates its expression,thereby enhancing the mitochondrial respiratory function of prostate cancer and promoting its proliferation and metastasis.