The Role And Mechanism Of FTO-mediated M~6A Modification In Diabetic Intimal Hyperplasia

Background Diabetes mellitus is known as an independent risk factor of intravascular restenosis after percutaneous coronary intervention(PCI)in coronary artery disease.Previous studies have shown that obesity-related protein(FTO)can affect diabetic intimal hyperplasia,but its molecular mechanism has not been fully clarified.Objective This study aims to explore the effect of FTO downregulation on the migration of vascular smooth muscle cells(VSMCs)by infecting lentivirus vectors interfered the expression of FTO into primary cultured rat VSMCs and vascular tissues of rats,thus providing a novel therapeutic target for the prevention and treatment of diabetic intima hyperplasia.Methods(1)In vitro:The primary cultured rat VSMCs were randomly divided into four groups:normal control group(NG group),high glucose group(HG group),negative lentivirus group(LV-NC-RNAi+HG group)and FTO down-regulated group(LV-Fto-RNAi+HG group).After infecting lentivirus with MOI=25 into VSMCs for 3 days,the lentivirus infection efficiency was observed through fluorescence microscope.The migration ability of VSMCs was evaluated by Transwell chamber test,Scratch test,and migration-related protein detection.The protein expression levels of FTO,LANCL2,AKT,and p-AKT were detected by Western blot.The m~6A methylation abundance of total RNA was detected by m~6A dot blot test.The m~6A methylation level of LANCL2 m RNA was detected by Me RIP-q PCR assay.(2)In vivo:Sixty healthy male SD rats were randomly divided Nor+Sham group,Nor+Injury group,DM+Injury group,DM+Injury+LV-NC-RNAi group,and DM+Injury+LV-Fto-RNAi group.The rats were fed with high-sugar and high-fat diet for 4 w,and after 3 days of single intrapitoneal injection of low-dose streptozotocin(STZ,20 mg/kg),random blood glucose of the rats was measured in the caudal vein to evaluate the modeling of diabetic rats(blood glucose≥15 mmol/L was considered as successful modeling).After 2 weeks of high-fat diet,carotid artery balloon injury operation was performed.After the operation,the common carotid artery and internal carotid artery were clipped by a bulldog clamp to temporarily close the lumen of the damaged common carotid artery segment when the external carotid artery was permanently ligated.Then the lentivirus or normal saline was injected into the damaged segment and infected for 20 min before restoring the blood flow,thus to establish the carotid ballon injury model of diabetic rats.Carotid artery balloon injury was observed in normal group after 6 weeks of general feeding,while no balloon injury was observed in Nor+Sham group after exposure of the common carotid artery,internal carotid artery and external carotid artery.Other steps were the same as those in operation group.H&E staining was used to detect intimal hyperplasia.The protein expression levels of FTO,LANCL2,AKT,p-AKT,and MMP9 were detected by Western blot.Results(1)In vitro:(1)In VSMCs treated with high glucose,the expression of FTO was up-regulated and the overall m~6A methylation abundance was decreased:compared with 0 h,the expression of FTO was most significantly increased at 48 h after high glucose stimulation of VSMC,and m~6A dot blot test showed that the overall m~6A methylation abundance was decreased in HG group when compared with NG group(P<0.05).(2)Successful lentivirus infection to rat primary VSMCs:Under fluorescence microscopy,the green positive VSMCs could be found in the LV-NC-RNAi+HG and LV-Fto-RNAi+HG groups but not in the Control group.(3)Down-regulation of FTO inhibited the high glucose-induced VSMCs migration:Western blot detection of migration-related proteins showed that compared with NG group,the protein level of MMP9 was significantly up-regulated in HG group;compared with LV-NC-RNAi+HG group,infection of LV-Fto-RNAi significantly down-regulated the protein level of MMP9(P<0.05).Transwell chamber test and Scratch test also showed that the number of migrated cells and mobility were significantly increased in the HG group,and FTO downregulation significantly inhibited the cell migration induced by hyperglycemia(P<0.05).(4)Down-regulation of FTO inhibits the activation of LANCL2/AKT signaling pathway in VSMCs:High glucose treatment promoted the expression of LANCL2 and subsequent ATK phosphorylation,and downregulation of FTO significantly inhibited the LANCL2/AKT signaling pathway induced by high glucose.This effect was observed to be reversed after the application of the LANCL2 agonist BT-11.(P<0.05).(5)Me RIP-q RCR assay showed that the abundance of m~6A methylation modification of LANCL2 m RNA was significantly increased after FTO downregulation(P<0.05).(2)In vivo:(1)The carotid balloon Injury model of rats was successfully constructed:compared with Nor+Sham group,the intima area and the intima/media area ratio were significantly increased in Nor+Injury group(P<0.05).(2)Downregulation of FTO can improve carotid intimal hyperplasia after balloon injury in diabetic rats:H&E staining results showed that compared with DM+Injury+LV-NC-RNAi group,the intima area and the ratio of intima/media area in DM+Injury+LV-Fto-RNAi group were significantly decreased(P<0.05).(3)Down-regulated FTO inhibited migration-related protein levels in carotid intima tissue of diabetic rats after balloon Injury:Compared with DM+Injury+LV-NC-RNAi group,the protein level of MMP9 in vascular tissue of DM+injury+LV-Fto-RNAi group was decreased(P<0.05).(4)Down-regulation of FTO inhibits the LANCL2/AKT signaling pathway in carotid intima tissue of diabetic rats after balloon Injury:compared with Nor+Injury group,the protein levels of LANCL2 and p-AKT in vascular tissue of DM+Injury group were increased,while the total AKT level was not significantly changed.Compared with DM+Injury+LV-NC-RNAi group,the protein levels of LANCL2 and P-Akt in vascular tissue of DM+Injury+LV-Fto-RNAi group were markedly decreased while the total AKT level was not significantly changed(P<0.05).Conclusion Down-regulation of FTO can promote the m6A methylation of LANCL2m RNA and reduce the expression of LANCL2,thus inhibiting the VSMC migration through the suppression of AKT signaling and driving an antagonistic role in diabetic intimal hyperplasia.