Effect of down-regulation of let-7c/g on triggering a double-negative feedback loop and promoting restenosis

Background:Excessive proliferation and migration of vascular smooth muscle cells(VSMCs)are the main causes of restenosis(RS)in diabetic lower extremity arterial disease(LEAD).However,the relevant pathogenic mechanisms are poorly understood.Methods:In this study,we introduced a“two-step injury protocol”rat RS model,which started with the induction of atherosclerosis(AS)and was followed by percutaneous transluminal angioplasty(PTA).Hematoxylin-eosin(HE)staining and immunohistochemistry staining were used to verify the form of RS.Two-step transfection was performed,with the first transfection of Lin28a followed by a second transfection of let-7c and let-7g,to explore the possible mechanism by which Lin28a exerted effects.5-ethynyl-2΄-deoxyuridine(EdU)and Transwell assay were performed to evaluate the ability of proliferation and migration of VSMCs.Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)were performed to detect the expression of Lin28a protein and let-7 family members.Results:Using a combination of in vitro and in vivo experiments,we discovered that let-7c,let-7g,and microRNA98(miR98)were downstream targets of Lin28a.More importantly,decreased expression of let-7c/let-7g increased Lin28a,leading to further inhibition of let-7c/let-7g.We also found an increased level of let-7d in the RS pathological condition,suggesting that it may function as a protective regulator of the Lin28a/let-7 loop by inhibiting the proliferation and migration of VSMCs.Conclusion:These findings indicated the presence of a double-negative feedback loop consisting of Lin28a and let-7c/let-7g,which may be responsible for the vicious behavior of VSMCs in RS.