The Role And Mechanism Of RNA M6A Methylation In Atherosclerosis

Background and objective:Coronary artery disease(CAD)is the main disease burden in China.With the update of etiology and pathophysiology of CAD and the advance of the treatment technology,the prognosis has obvious improvement,however,its prevalence has remained high for many years.Its etiology,pathogenesis and new therapeutic targets need to be further explored.As a common post-transcriptional modification,RNA methylation has received much attention in recent years.The present study aims to explore the role of RNA m6A methylation in the development of CAD.Methods:1.A total of 56 CAD patients and 30 healthy controls were randomly selected,the m6A level of total RNA from peripheral blood mononuclear cells was detected by colorimetry.Among them,5 patients and 5 controls were randomly selected to describe the CAD RNA m6A modification pattern using methylated RNA immunoprecipitation-sequencing(MeRIP-seq)and bioinformatics analyses.2.Gain-and loss-of-function strategy was used to explore the influence of fat and obesity associated(FTO)gene on human aortic smooth muscle cells(HASMCs)proliferation and migration.Overexpression of the FTO gene in HASMCs,MeRIP-seq,bioinformatics analysis,Western blot and phenotypic recovery experiments were used to clarify the molecular mechanism of FTO in cell phenotype.3.The effect of FTO on the intimal hyperplasia of carotid artery after balloon injury were evaluated in Sprague-Dawley(SD)rats.Results:1.RNA methylation level(m6A%)of peripheral blood mononuclear cell in CAD patients was lower than that in controls(0.21%vs.0.25%,P<0.001).A total of 24625 methylation sites were detected by MeRIP-seq.Of those,9038 m6A methylation sites showed quantitative differences between the two groups.GO and KEGG pathway analyses showed that differential m6A sites were significantly related with cell proliferation,migration and apoptosis.2.In HASMCs,overexpression of the FTO gene inhibits migration of these cells,in contrast,knockdown of the FTO gene promotes migration.However,neither overexpression nor knockdown of the FTO gene showed distinct effect on HASMCs proliferation.Treating cells with ENT alleviates the inhibitory effect of the FTO gene on cell migration,showing that inhibiting effect of the FTO gene on cell migration is dependent on its m6A demethylase activity.FTO was shown to regulate the m6A methylation level and expression level of the COL8A1 gene using MeRIP-seq and RNA-seq.Western blot experiments confirmed that the COL8A protein level was regulated by FTO.Migration phenotype recovery experiments supported the notion that the COL8A1 gene is involved in the regulation of the FTO gene on HASMCs migration.3.In SD rats,overexpression of the FTO gene at the vascular injury site significantly inhibited the intimal hyperplasia of carotid artery after balloon injury.The intima/media area ratio was significantly reduced.Knockdown of the FTO gene at the site of vascular injury significantly promotes the intimal hyperplasia of carotid artery with the increased the intima/media area ratio.Conclusions:1.This study depicts the RNA m6A modification map of CAD.CAD was associated with the lower RNA m6A levels.Differential m6A sites were associated with cell proliferation,migration,and apoptosis.2.FTO regulated the expression of COL8A1 gene through influencing its mRNA m6A methylation level and then inhibited HASMCs migration.3.FTO was confirmed to inhibit intimal hyperplasia of carotid artery after balloon injury in rat.