The Function And Mechanism Of Let-7c In Restenosis After Percutaneous Transluminal Angioplasty Of Diabetic Lower Extremity Arterial Disease

BackgroundDiabetes lower extremity arterial disease(DLEAD)is one of the common chronic complications of type 2 diabetes.As the incidence of diabetes has increased in recent years,the incidence rate of DLEAD is also rising year by year.The pathological range of DLEAD mainly focuses on the branches of the arteries and small blood vessels below the knee,and the condition progresses rapidly.Therefore,conservative medical treatment is not effective.The special lesion site makes percutaneous transluminal angioplasty(PTA)become the main clinical treatment method for DLEAD.However,the high incidence of restenosis after PTA treatment for DLEAD greatly limits its clinical efficacy.Therefore,effective prevention and treatment of restenosis after PTA is the key to improving the efficacy of DLEAD treatment,and it is also an urgent key issue that needs to be addressed.Our previous research found that compared with atherosclerosis,the proliferation and migration of vascular smooth muscle cells(VSMCs)in restenosis were significantly enhanced,and further confirmed that Lin28a/let-7 can effectively regulate the migration and proliferation of VSMCs.This study further explores based on previous research findings.The let-7 miRNA family is usually highly expressed in differentiated cells,with highly conserved sequences and functions,involved in regulating cell proliferation,differentiation,and other life processes.As a member of the let-7 family,let-7c is crucial for cell growth and proliferation.Transforming growth factor β(TGF-β)is a multifunctional cytokine that starts the downstream signal pathway by binding with the TGF-β receptor type Ⅰ(TGFBR1)and TGF-β receptor type Ⅱ(TGFBR2),phosphorylates its downstream signal molecule SMAD protein,and finally regulates gene expression.This study used miRWalk and miRDB databases to predict that let-7c has a binding site to the 3’ untranslated region(3’UTR)of TGFBR1.Previous studies have pointed out that let-7c is involved in the development and progression of renal fibrosis through the regulation of the TGF-β signaling pathway.However,its role in the proliferation and migration of VSMCs in the restenosis of DLEAD after PTA has not been reported.This study started with let-7c to explore its potential mechanism in regulating the proliferation and migration of VSMCs in RS,and to provide a new idea for the prevention and treatment of restenosis after PTA treatment of DLEAD.Objectives1.Through establishing the rat model of type 2 diabetes restenosis to clarify that let-7c and TGFBR1 are involved in the occurrence and development of restenosis.2.By regulating the expression of let-7c and TGFBR1 in VSMCs,to explore the specific molecular mechanism of let-7c regulating TGFBR1 to regulate the proliferation and migration of VSMCs in restenosis.Materials and Methods1.The AS model of type 2 diabetes rats was established by balloon dilation and strain surgery.The RS model of type 2 diabetes rats was established by "double injury" operation,namely balloon dilatation and strain surgery and balloon dilatation surgery.Use animal ultrasound to verify whether the model has been successfully constructed,and collect materials from the successfully modeled rats.2.Using quantitative real-time(qRT-PCR)to detect the expression level of let-7c in AS and RS.3.The stripping method was used to obtain primary VSMCs,and VSMCs were transfected with let-7c mimics and let-7c inhibitor respectively.Cell Counting Kit 8(CCK-8)and EdU cell proliferation assay(EdU)were used to detect the proliferation ability of VSMCs;Transwell migration assay(Transwell)was used to detect the migration ability of VSMCs..4.Using bioinformatics databases to predict downstream targets of let-7c and screen for potential downstream target gene TGFBR1.VSMCs were transfected with let-7c mimics and let-7c inhibitor respectively.qRT-PCR was used to detect the mRNA expression level of TGFBR1 in VSMCs;Western blot was used to detect the protein expression levels of TGFBR1 and p-SMAD3 in VSMCs.5.qRT-PCR was used to detect the mRNA expression level of TGFBR1 in AS and RS.6.VSMCs were transfected with TGFBR1 plasmid(OE.TGFBR1)and TGFBR1 small interfering RNA(si.TGFBR1)respectively.CCK-8 and EdU were used to detect the proliferation ability of VSMCs;Transwell was used to detect the migration ability of VSMCs;qRT-PCR was used to detect the mRNA expression level of TGFBR1 in VSMCs;Western blot was used to detect the protein expression levels of TGFBR1 and p-SMAD3 in VSMCs.7.VSMCs were co-transfected with let-7c mimics+OE.TGFBR1 and let-7c inhibitor+si.TGFBR1 respectively.CCK-8 and EdU were used to detect the proliferation ability of VSMCs;Transwell was used to detect the migration ability of VSMCs;qRT-PCR was used to detect the mRNA expression level of TGFBR1 in VSMCs;Western blot was used to detect the protein expression levels of TGFBR1 and p-SMAD3 in VSMCs.Results1.The qRT-PCR results indicated that let-7c was downregulated in RS compared to AS,indicating that let-7c may be involved in the formation and progression of restenosis.2.The results of CCK-8 and EdU showed that upregulation of let-7c inhibited the proliferation of VSMCs,while downregulation of let-7c promoted the proliferation of VSMCs.The Transwell results indicated that upregulation of let-7c inhibited the migration of VSMCs,while downregulation of let-7c promoted the migration of VSMCs.It has been demonstrated that let-7c negatively regulates the migration and proliferation of VSMCs.3.The results of qRT-PCR and Western blot showed that upregulation of let-7c reduced the mRNA and protein expression levels of TGFBR1,as well as the protein expression level of p-SMAD3;Downregulation of let-7c increased the mRNA and protein expression levels of TGFBR1,as well as the protein expression level of p-SMAD3.It has been demonstrated that let-7c negatively regulates the expression of TGFBR1 and p-SMAD3 in VSMCs.4.The qRT-PCR results indicated that TGFBR1 was upregulated in RS compared to AS,indicating that TGFBR1 may be involved in the formation and progression of restenosis.5.The results of CCK-8 and EdU showed that upregulation of TGFBR1 promoted the proliferation of VSMCs,while downregulation of TGFBR1 inhibited the proliferation of VSMCs.The Transwell results indicated that upregulation of TGFBR1 promoted the migration of VSMCs,while downregulation of TGFBR1 inhibited the migration of VSMCs.It has been demonstrated that TGFBR1 positively regulates the migration and proliferation of VSMCs.6.Western blot results showed that upregulation of TGFBR1 increased the protein expression level of p-SMAD3,while downregulation of TGFBR1 decreased the protein expression level of p-SMAD3.It has been demonstrated that TGFBR1 positively regulates the expression of p-SMAD3 in VSMCs.7.The rescue experiment results showed that overexpressing both let-7c and TGFBR1 can increase the expression level of p-SMAD3,and partially reverse the migration and proliferation of VSMCs inhibited by let-7c overexpression;In addition,silencing both let-7c and TGFBR1 can reduce the expression level of p-SMAD3,and partially reverse the migration and proliferation of VSMCs promoted by let-7c silencing.It has been confirmed that let-7c can regulate the migration and proliferation of VSMCs through the TGFBR1/SMAD3 pathway in VSMCs.Conclusion1.The reduced expression of let-7c and the increased expression of TGFBR1 in RS suggest that let-7c and TGFBR1 are involved in the development of restenosis after PTA in DLEAD.2.Let-7c negatively regulates the migration and proliferation of VSMCs,while TGFBR1 positively regulates the migration and proliferation of VSMCs.3.Let-7c inhibits migration and proliferation of VSMCs by negatively regulating TGFBR1/SMAD3 pathway,and plays a protective role in restenosis after PTA in DLEAD.