Effects Of Mm9_circ₀10056 On Diabetes-induced Endothelial To Mesenchymal Transition And Its Mechanism

Aims:To observe the effect of mm9_circ₀10056 on diabetes-induced endothelial to mesenchymal transition（EndoMT）,and to explore its possible molecular mechanism.Methods:Total RNA was treated with ribonuclease（RNase）R to detect cyclic characteristic of mm9_circ₀10056;Fluorescence in situhybridization（FISH）was used to detect the Intracellular localization of mm9_circ₀10056.Thoracic aortas were obtained from Spontaneous type II diabetes model db/db mice（8 weeks old,12 weeks old,n=9）and wild type（WT）mice（8 weeks old,12 weeks old,n=9）,and the relative expression of mm9_circ₀10056 and the occurrence of EndoMT were detected in endothelial layer.Mouse aortic endothelial cells（MAEC）was cultured and treated with advanced glycation end products（AGEs）（0,50μg/ml,100μg/ml,200μg/ml）for 4h,24 h and 48 h and its effect on the mm9_circ₀10056 expression was observed.Adenovirus mediated overexpression of mm9_circ₀10056 and siRNA mediated knockdown of mm9_circ₀10056 in MAEC induced by AGEs,and the effect of mm9_circ₀10056 on AGEs-induced EndoMT was observed.Western blotting and immunofluorescence assays were used to evaluate the occurrence of EndoMT.RT-qPCR was used to detect the mRNA expression of mm9_circ₀10056 and Ube3 a,and Western blotting was used to detect Ube3 a protein levels.Results:Mm9_circ₀10056 generated from Ube3 a gene;mm9_circ₀10056 was resistant to ribonuclease because of its high stability,and expressed in the cytoplasm and nucleus.Compared to WT group,mm9_circ₀10056 expression was down-regulated in different aging db/db mice aortic endothelial layer and EndoMT was observed there.AGEs could concentration-dependently and time-dependently suppress mm9_circ₀10056 expression;Overexpression of mm9_circ₀10056 in the MAEC restored the AGEs-induced decrease in CD31 levels and the increase in α-SMA levels,however,mm9_circ₀10056 knockdown can induce EndoMT.Up-regulation and down-regulation of mm9_circ₀10056 could regulate the expression of Ube3 a in MAEC.Ube3 a expression is down-regulated in both the diabetic mouse aortic endothelial layer and MAEC induced by AGEs;Overexpression of Ube3 a in the MAEC restored the AGEs induced EndoMT.Conclusion:Mm9_circ₀10056 is involved in the process of diabetes-induced EndoMT in the aortic endothelial cells,and its mechanism may be related to mm9_circ₀10056/Ube3 a pathway.