Effect Of MiR-375Overexpression On AGEs-mediated Diabetic Vascular Cells

The incidence of diabetes mellitus(DM) is increasing year by year and its mortality ranks next to cardiovascular and cerebrovascular disease. Diabetic vascular injury is one of the main complications leading to morbidity and mortality. NuMerous evidence demonstrates that advanced glycation end products (AGEs) plays a pivotal role in the development and progression of diabetic vascular injury. AGEs induces massive ROS generation which activates protein kinase C and NF-κB via intracellular signaling cascade. Subsequently, the molecular pathway of diabetic complication was initiated, causing elevation of profilin1which is the marker of diabetic vascular injury, redistribution of cytoskeletal reorganization. Finally, cell apoptosis was induced as a result of increased cell permeability.MiR-375was recently characterized as a new mircroRNA and was at first found in pancreas. Its expression is greatly correlated with the morpHology, development and function of pancreas. It has many target proteins. miR-375modulates insulin secretion through binding to MTPN(MyotropHin). Several studies have demonstrated that Mtpn is an intracellular NF-κB activating factor, and miR-375can alter the activity of NF-B by regulating Mtpn. In this study, miR-375expression vector was designed and constructed and subsequently miR-375expression plasmid was transfected into injured vascular cells. Meanwhile, four groups for further study respectively named Huvecl2as control, injured vascular cells, vascular cells after injury inhibition and cells with miR-375overexpression. Cells were harvested24hours after treatments. The expression of Mtpn, NFκB, profilinl and sICAMl were measured in both mRNA and protein level. F-actin was examined via fluorescence staining. Cell apoptosis was assessed by flow cytometry. The result showed that in injured vascular cells with miR-375overexpression, the mRNA and protein expression of targeted gene Mtpn were declined,The reduction of NFκB activity, downregulation of the diabetic vascular injury marker profilinl, restoration of F-actin expression, a decrease of sICAMl expression and reduced cell apoptosis were also observed.The results this study provides preliminary evidence that miR-375may inhibit the injuries of vascular cells mediated by AGEs-mediated diabetes and may become a new target for gene therapy to treat vascular complications caused by diabetes.