| Objective To explore the effect and potential mechanism of miR-24 on intimal hyperplasia after vascular injury in diabetes using adenovirus vectors overexpressing miR-24 in vitro and in vivo.Methods(1)The VSMCs were randomly divided into four groups: NG group,HG group,Ad-Scramble+HG group,and Ad-miR-24+HG group.In addition to adenovirus transfection for 3 days in Ad-Scramble+HG group and Ad-miR-24+HG group,all VSMCs were stimulated with glucose at concentration of 5 mmol/L in NG group and 30 mmol/L in HG group for 16 h.(2)The transfection efficiency of adenovirus in VSMCs was evaluated by fluorescence microscopy.(3)CCK-8 assay was used to detect VSMCs proliferation.(4)Scratch test and Transwell chamber test were used to detect VSMCs migration.(5)Male SD rats were fed with high-sugar and high-fat diet for 4 weeks,followed with low-dose STZ introperitoneal injection,to establish diabetic model.The carotid artery balloon injury was performed after 2 weeks of blood sugar stabilization.The injured segments were locally transfected with corresponding virus vectors for 20 min before blood flow restoration.(6)The transfection efficiency of adenovirus in injured vascular tissues was observed under fluorescence microscopy.(7)HE staining was used to evaluate intimal hyperplasia.(8)The expression of PCNA was detected by immunohistochemistry.(9)Masson staining was used to detect fibrosis in injured vascular tissue.(10)qRT-PCR was used to detect the expression level of miR-24 and PIK3R1 mRNA.Western blot was used to detect the protein level of PI3 K p85α,Akt,p-Akt,mTOR,p-mTOR,4E-BP1,p-4E-BP1,p70s6 k,p-p70s6 k,MMP 2,MMP 9,collagen Ⅰ,as well as collagen Ⅲ.Results(1)Adenovirus were successfully transfected into VSMCs.VSMCs in Ad-Scramble+HG and Ad-miR-24+HG group performed obvious green fluorescence under fluorescence microscope.(2)As the results of CCK-8 test shown,up-regulation of miR-24 inhibited VSMCs proliferation induced by HG(P<0.05).(3)Up-regulation of miR-24 inhibited VSMCs migration and migration-related protein(MMP 2,MMP 9)expression in HG-induced VSMCs(P<0.05).(4)Up-regulation of miR-24 significantly inhibited the expression of Collagen I and Collagen III induced by HG(P<0.05).(5)Under stimulation with HG for 16 h,the expression of miR-24 in VSMCs decreased significantly,while the expression of PIK3R1 mRNA increased greatly(P<0.05).After transfection with Ad-miR-24,the expression of miR-24 increased obviously(P<0.05),consisted with a great decrease of PIK3R1(P<0.05).(6)Up-regulation of miR-24 inhibited the expression and activation of PI3K/Akt signaling pathway-related molecules in VSMCs under HG stimulation(P<0.05).PI3 K p85 agonist 740Y-P could weaken the effects of miR-24.(7)Adenovirus vectors were successfully transfected into the common carotid artery in diabetic rats.Obvious green fluorescence was found in Ad-Scramble and Ad-miR-24 group under fluorescence microscope.(8)Up-regulation of miR-24 could inhibit intimal hyperplasia.Compared with Ad-Scramble group,both intimal area and ratio of intimal area to medial area in Ad-miR-24 group were significantly reduced(both P<0.05).Meanwhile,up-regulation of miR-24 significantly inhibited the expression of migration-related protein(P<0.05).(9)The results of vascular immunohistochemistry showed that the ratio of cells positive to PCNA in Ad-miR-24 group was significantly lower than that in Ad-Scramble group(P<0.05).(10)Compared with Ad-Scramble group,up-regulation of miR-24 significantly inhibited the formation of vascular collagen both in morphological and protein analysis(P<0.05).(11)The expression of miR-24 in diabetic arteries was decreased significantly(P<0.05),while PIK3R1 mRNA was increased obviously(P<0.05)when compared to the non-diabetic.Ad-miR-24 induced an overexpression of miR-24 and a decline of PIK3R1 mRNA in vivo(P<0.05).(12)Up-regulation of miR-24 could inhibit the activation of PI3K/Akt signaling pathway in injured vessels(P<0.05).Conclusion miR-24 up-regulation attenuates intimal hyperplasia after vascular injury in diabetic rats through inactivation on PI3K/Akt signaling transduction. |
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