The Function And Mechanism Of Lin28a In Diabetic Lower Extremity A Rterial Disease After Percutaneous Transluminal Angioplasty (PTA)

ObjectivesTo investigate the high expression of Lin28a in diabetic lower extremity arterial disease after percutaneous transluminal angioplasty(PTA)by establishing a restenosis rat model,and to investigate the mechanism by cell experiment.Materials and methodsPart one:One-month-old male SD rats(90-100g)were obtained.The rats adapted to the new environment for at least 7 days,and then they were fed with a high-fat diet.After 3 weeks,we performed intraperitoneal insulin tolerance test(IPITT)and intraperitoneal glucose tolerance test(IPGTT)to detect whether the rats with insulin resistance.Type 2 diabetes mellitus was induced by intraperitoneal injection of streptozocin(STZ)to rats with insulin resistance.Three days after STZ injection,rats with fasting blood glucose(FBG)? 11.1 mmol/L in 3 consecutive analyses were considered as the diabetic rat model.Then the diabetic rats were devided into two groups:atherosclerosis(AS)and restenosis(RS).The AS group were given balloon-induced endothelial injury surgery to establish atherosclerotic vessel.The RS group were given the same operation as the AS group during the first surgery.After 4 weeks' high-fat diet,PTA was taken if atherosclerotic damage was formed in the iliac artery to induce restenotic plaque.Finally,the rats were received ultrasound examination to detect the formation of plaque.Hematoxylin-eosin(HE)was taken to assess the neointimal hyperplasia.Immunohistochemical analysis was performed to assess the expressions of Lin28a and a-SMA in two groups.Immunofluorescent double staining was used to detect the co-expression of Lin28a and ?-SMA.The RNA levels of Lin28a in two groups were detected by qRT-PCR.Part two:VSMCs in primary culture were obtained by using explant culture method.The cells were divided into three groups:group A:over-expression lentiviral vector Lin28a,group B:the empty lentiviral vector(control group),group C:the lentiviral vector containing Lin28ashRNA.We detect the expression of Lin28a by using western blot and qRT-PCR.Cell proliferation was detected by EdU method and cell migration was detected by transwell method.Statistical analysis:We presented all data as mean± Standard Error(SE).We performed comparisons by using one-way ANOVA with SPSS Software(Version 22.0,SPSS China,Shanghai,China).We considered that P<0.05 was statistically significant.ResultsPart one:1.Animals:The experiment was implemented using 40 SD rats.After being injected with STZ,30 rats generated hyperglycemia.At the end of the experiment,there were 8 rats in AS group,and 7 rats in RS group.Body weights and levels of blood glucose were shown in Table 1.There were no significant differences between AS and RS group in baseline.2.Color doppler ultrasonogram flow imaging:Once diabetes was diagnosed,ultrasonography was used to show the condition of the vessels in different time-course.Fig.1B showed that after balloon injury surgery atherosclerosis plaques were formed.After PTA,ultrasound was used to detect the recanalization of artery stenosis(Fig.1C).In the RS group,after PTA surgery,stenosed iliac arteries was recanalized(Fig.1D).3.Characterization of atherosclerotic and restenotic plaques:Samples were collected from the iliac artery of rats undergoing atherosclerotic or restenotic lesions.HE showed that atherosclerotic and restenotic plaque were formed(Fig.2).Immunohistochemical analysis revealed a significantly higher expression of a-SMA(a marker for VSMCs)in restenotic plaques when compared to atherosclerotic plaques.Immunofluorescent double staining showed that the a-SMA in all visualized Lin28a positive cells was positive.4.qRT-PCR showed that the expression of Lin28a in RS group was higher than that in AS group.Part two:1.Cultured primary VSMCs,and immunofluorescence demonstrated that a-SMA and SM-22 the positive in 95%cells.2.Transfected VSMCs by using over-expression lentiviral vetor Lin28a(group A)?the empty lentiviral vetor(group B)and lentiviral vector containing Lin28ashRNA(group C).3.Western blotting and qRT-PCR analysis showed that the expression of Lin28a in group A was increased,and group C was decreased compared with group B.4.EdU method showed that the proliferation of VSMCs in group A was increased,group C was decreased.5.Transwell showed that the migration of VSMCs in group A was increased,group C was decreased.Conclusions1.Our restenosis rat model proved that plaques comprised of large number of VSMCs in restenosis artery.Numbers of lin28a-positive cells in restenotic plaques were higher than that in atherosclerotic plaques,and the a-SMA in all visualized Lin28a positive cells was a-SMA positive.2.In vitro experiment:a significant decrease in cell proliferation and migration was found by downregulating Lin28a in VSMCs with an shRNA.