| BackgroundAbdominal aortic aneurysms(AAAs),a devastating disease with high morbidity and whose mortality rate excesses over 80%once rupture despite advancing surgery.It is consensus that deferring development of AAA is key to curb the AAA catastrophic consequences.In clinic,lifestyle and metabolic improvements such as smoking cessation,blood pressure,lipid and glucose control are conventional measures to retard AAA development.Controlling these risk factors do obtain some benefits,however,the AAA morbidity still remains high.Blocking the pathogenesis of AAA formation may be deemed as a potential strategy to preventing or deferring AAA development,and has been gaining increasing zest in the endeavor to address AAA issue.N6-methyladenosine(M6A)is regarded as the most common post-translational modification for mRNA and a general one for ncRNA in eukaryotic organizations by mediating over 80%of RNA methylation.N6-methyladenosine of RNA exists within disease pathophysiological processes through promoting pre-RNA spicing,mRNA translation,and enhancing RNA stability.Present studies reveal that M6A RNA post-translational modification play a key role in diverse pathophysiological processes,such as cancer cells growth,acute myelogenous leukemia,embryonic stem cell growth and differentiation and T cells homeostasis.Importantly,recent study also strengthened the critical roles M6A RNA post-translational modification has in cardiovascular disorders,such as myocardial ischemia and cardiomyocytes regeneration.Interestingly,Our preliminary experiments found that the RNA N6-methyladenosinemethylation levels was upregulated in AAA samples and that this upregulation was induced by elevated M6A methyl-transferases METTL3.Those findings evoke a notion that whether N6-methyladenosine of RNA participate in the progression of AAA,which was implied by other studies.Rencet studies reported that METTL3 increased the expression of inflammatory cytokines and activated the NF-kB signalling pathway as well as the MAPK signaling pathway,which implicated in AAA development.Moreover,METTL3 potentiates multiple pri-miRNAs splicing to form mature miRNAs,which are regarded crucial and upregulated in aortic aneurysm formation.Taken together,we hypothesized that METTL3 facilitates pri-miRNAs maturation by promoting their M6A RNA modification,mediating vascular inflammation development and thus participates in AAA formation.In line with this hypothesis,we detected the N6-methyladenosine levels of RNA and METTL3 in AAA samples.Following,the effects of METTL3 on AAA development was explored in two AAA models.Mechanically,we revealed that the signaling pathway under these effects is METTL3-mediated miR-34a maturation by pri-miR-34a M6A RNA modification.Of crucial importance,to illustrate that miR-34a acts as an intermediary in METTL3-mediated AAA development,we also overexpressed and knocked downed miR-34a to explore its role in AAA and the underlying mechanism.Methods and ResultsGain-and loss-of-function experimentsAdeno-associated virus(AAV)carrying METTL3 siRNA(Sh-METTL3),overexpression plasmid(AAV-METTL3)and their control viruses(Scr-RNA and AAV-GFP)were constructed.Sh-METTL3,AAV-METTL3,and their control viruses were respectively injected into ApoE-/-mice or C57BL/6J by tail vein.The METTL3-deleted mice and METTL3-overexpression mice were randomly selected to start receiving four weeks of Ang ? infusion or cacl2 treatment.Van Gieson staining of elastin(EVG)?Immunohistochemical(IHC)?RT-qPCR and Western Bolting and other experimental methods to clarify the role of METTL3 in AAA and relevant vascular pathological changes?Results:(1)After 28 days of Ang ? infusion,there was significant decreasing of AAA formation in the Sh-METTL3 group compared with that in the Scr-RNA group.Compared with Scr-RNA group,the maximal abdominal aortic diameter,and the elastin degradation score were remarkably reduced in Ang ?-treated sh-METTL3 ApoE-/-mice.(2)Immunohistochemical results shown that knock down the METTL3 remarkably attenuated the levels of vascular macrophages infiltration,MMP2 expression,MCP-1/CCL2 expression,and P21 expression in Ang ? treated ApoE-/?mice.(3)Western Bolting results shown that METTL3 deficiency remarkably reduced the levels of MMP2 expression,MCP1/CCL2 expression,and P21 expression in Ang II treated ApoE-/-mice,while overexpression METTL3 showed the opposite effects.(5)Similar trends of function experiments upon METTL3 dysregulation were obtained in CaC12-induced AAA model.Mechanism experiments(1)MeRIP-qPCR results demonstrated that M6A level of pri-miR-34a was increasing in AAAs or overexpression METTL3 VSMC,while that of METTL3 deficiency VSMC was decreasing.(2)Pri-miR-34a was increased in METTL3-depleted cells and decreased in METTL3-overexpressing cells.Compared with pri-miR-34a,mature miR-34a exhibited contrast expression in METTL3-knockdown or METTL3-overexpressing cells.(3)We also observed an increased pri-miR-34a bound to DGCR8 in METTL3-overexpressing cells when we immunoprecipitated DGCR8 from those cells,and we measured these pri-miR-34a by qRT-PCRConclusionMETTL3 promoted AAA formation by accelerating the pri-miR-34a process in a DGCR8-dependent manner and inhibited the expression of SIRT1,the target gene of miR-34a?... |
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