| Objective The major aim of the present work was to observe the influence of Micro RNA-24(miR-24) on intimal hyperplasia in diabetic rats after carotid artery injury and explore the molecular mechanistic.Methods Sixty male SD rats weighing 170 g were used for the current study. The rats were fed a high-fat diet(20% lard stearin, 10% sucrose, and 0.1% bile salt were added to the normal diet) for four weeks followed by intraperitoneal injection with streptozotocin(STZ, 45 mg/kg) for another week. Diabetes was considered to be induced once hyperglycemia became evident(blood glucose levels after 12 h of fasting ≥16.7 mmol/L).The 48 STZ-induced diabetic rats were randomly and equally assigned into four groups(n=12): Sham group, carotid artery balloon injury with phosphate buffered saline treatment group(PBS group), carotid artery balloon injury transfected with adenovirus expressing Scramble-GFP group(Ad-Scramble group), and carotid artery balloon injury transfected with adenovirus expressing miR-24-GFP group(Ad-miR-24 group). Two weeks after adenovirus or PBS transduction, the rats were euthanized and the tissues were harvested for detection. Adenovirus transfection efficiency was evidenced by green fluorescence under fluorescence microscopy. The level of neointimal hyperplasia was examined by hematoxylin-eosin(HE) staining. Vascular smooth muscle cells(VSMCs) proliferation in the neointima was evaluated by immunostaining for proliferating cell nuclear antigen(PCNA). The m RNA levels of miR-24 and cell cycle-associated molecules(Cyclin D1, p21)were determined by quantitative real-time PCR(q RT-PCR) analysis. PCNA, Wnt4,Disheveled-1(Dvl-1), β-catenin, Cyclin D1 and p21 protein levels were measured by western blotting analysis.Results(1) Fasting blood glucose levels started to be increased at the third day after the injection of STZ, and the level continued to be significantly higher in diabetic rats until seven days compared with the normal rat group(P<0.05).(2) After Ad-miR-24 was transfected into carotid artery in diabetic rats fourteen days later, GFP was detected by immunofluorescence and adenoviral transfection markedly increased miR-24 expression.(3) Compared with Sham group, PBS and Ad-Scramble transfection group had a severe intimal hyperplasia(P <0.05). Ad-miR-24 treatment significantly reduced the neointimal area and intima/media(I/M) ratio compared with Ad-Scramble and PBS group(P<0.05).No significant differences in the medial area were observed between any of the groups(P>0.05).(4) Compared with Ad-Scramble and PBS groups, it could induce a significant reduction in the percentage of PCNA-positive cells in the neointima after treatment with Ad-miR-24(P<0.05). In addition, the protein level of PCNA using western blotting was consistent with the results of immunostaining.(5) Compared with Sham group, the protein levels of Wnt4, Dvl-1, β-catenin, were strikingly up-regulated in the carotid arteries of diabetic rats after balloon injury(P<0.05). Ad-miR-24 group could significantly reduce the levels of these above proteins compared with Ad-Scramble and PBS groups(P<0.05).(6)Compared with Sham group, both gene and protein expression levels of Cyclin D1 were increased, but p21 was repressed(P<0.05). Interestingly, over-expression of miR-24 significantly inhibited the gene and protein levels of Cyclin D1 but increased the levels of p21(P<0.05).Conclusion(1) Transfection with Ad-miR-24 markedly increased miR-24 expression in balloon injured carotid artery in the diabetic rats.(2) Over-expression of miR-24 could attenuate VSMCs proliferation and neointimal hyperplasia after vascular injury in diabetic rats, which is possibly related to the regulation of the expression of Cyclin D1 and p21 through the Wnt4/Dvl-1/β-catenin signaling pathway. |
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