miR-181b通过靶向PTEN调控缺血性脑卒中后血管新生的作用和机制研究

Effect and mechanism of miR-181b in regulating angiogenesis after ischemic stroke via targeting

目的探讨miR-181b对缺血性脑卒中后血管生成的影响及机制。方法选择SPF级雄性SD大鼠65只,鼠龄10~12周,体质量50~280 g。随机分为4组,即假手术组、模型组、miR-NC组和miR-181b组。除假手术组外,其他3组采用大脑中动脉线栓法建模,miR-NC组和miR-181b组大鼠分别尾静脉注射miR-NC及miR-181b质粒。治疗2周后,评价大鼠改良神经功能缺损评分(mNSS)。进行脑组织2,3,5-氯化三苯基四氮唑(TTC)染色,免疫荧光检测脑组织簇分化抗原34(CD34)、血管内皮生长因子(VEGF)表达。对人脐静脉血管内皮细胞(HUVEC)进行转染,分为Control组(正常培养)、miR-NC组(转染miR-NC空质粒)及miR-181b组(转染miR-181b mimics质粒)。CCK-8检测细胞增殖能力。划痕实验检测细胞迁移能力。成管实验检测成管能力。荧光素酶报告实验检测miR-181b与磷酸酯酶-张力蛋白同源物基因(PTEN)的靶向关系。实时荧光定量PCR(qRT-PCR)检测细胞、组织miR-181b表达水平。Western blot检测细胞、组织中PTEN蛋白表达水平。结果与假手术组比较,模型组大鼠mNSS、脑梗死面积升高(t=17.420,34.000,P<0.05)。与miR-NC组比较,miR-181b组大鼠mNSS、脑梗死面积降低(t=14.730,21.160,P<0.05)。与假手术组比较,模型组大鼠脑组织中miR-181b表达(0.14±0.02),CD34阳性细胞率(45.03%±5.22%),VEGF阳性细胞率(25.06%±5.11%),CD34、VEGF蛋白表达水平(0.25±0.06、0.21±0.04)下降,PTEN蛋白表达水平(0.93±0.05)升高(P<0.05)。与miRNC组比较,miR-181b组大鼠脑组织中miR-181b表达(3.02±0.45),CD34阳性细胞率(68.73%±7.04%),VEGF阳性细胞率(0.23%±0.06%),CD34、VEGF蛋白表达水平(0.86±0.12、0.72±0.09)升高,PTEN蛋白表达水平(0.37±0.02)下降(P<0.05)。miR-181b组细胞miR-181b表达水平,细胞24 h、48 h及72 h OD值、迁移率,节段长度、分支长度、连接数和网孔数高于miR-NC组(P<0.05)。miR-181b可负性调控PTEN表达。结论过表达miR-181b可靶向PTEN改善脑缺血再灌注损伤大鼠的神经功能,促进血管新生。

Objective To investigate the effect and mechanism of miR-181b on angiogenesis after ischemic stroke.Methods A total of 65 SPF grade male SD rats were sacrificed,with age of 10-12 weeks and body mass of 50-280 g.All of the rats were randomly divided into 4 groups:sham group,model group,miR-NC group and miR-181b group.Except control group,other three groups were modeled by middle cerebral artery occlusion method,the rats in miR-NC group and miR-181b group were injected with miR-NC and miR-181b plasmid through tail vein,respectively.After treatment for 2 weeks,the modified neurological severity score(mNSS)was evaluated.The expression of cluster differentiation antigen 34(CD34)and vascular endothelial growth factor(VEGF)in brain tissue was detected by immunofluorescence.The human umbilical vein endothelial cell(HUVEC)were transfected and divided into control group(normal culture),miR-NC group(transfected miR-NC empty plasmid)and miR-181b group(transfected miR-181b mimics plasmid).The cell proliferation ability was detected by cell counting kit-8(CCK-8)assay;cell migration ability was detected by scratch assay;tube formation assay was used to detect tube formation aility;the targeting relationship between miR-181b and human tensin homologue gene(PTEN)was detected by luciferase reporter assay;quantitative real-time fluorescence polymerase chain reaction(qRT-PCR)was used to detect expression level of miR-181b in cells and tissues;Western blot was used to detect expression levels of PTEN,CD34 and VEGF protein in cells and tissues.Resulsts Compared with sham group,mNSS and cerebral infarction area of model group were increased(t=17.420,34.000,P<0.05).Compared with miR-NC group,mNSS and cerebral infarction area in miR-181b group were decreased(t=14.730,21.160,P<0.05).Compared with sham group,the expression of miR-181b(0.14±0.02),CD34 positive rate(45.03%±5.22%),VEGF positive rate(25.06%±5.11%),CD34 and VEGF protein expression levels(0.25±0.06,0.21±0.04)in brain tissue of model group were decreased,and PTEN protein expression level(0.93±0.05)increased(P<0.05).Compared with miRNC group,miR-181b expression(3.02±0.45),CD34 positive rate(68.73%±7.04%),VEGF positive rate(0.23%±0.06%),CD34 and VECF protein expression levels(0.86±0.12,0.72±0.09)in brain tissue of miR-181b group were increased,while PTEN protein expression level(0.37±0.02)decreased(P<0.05).MiR-181b negatively regulated PTEN expression.The expression level of miR-181b,optical density(OD)value,migration rate,segment length,branch length,connection number and meshes of cells at 24-hour,48-hour and 72-hour in miR-181b group were higher than those in miR-NC group(P<0.05).The miR-181b negatively regulated PTEN expression.Conclusion It is demonstrated that overexpression of miR-181b could target PTEN to improve neural function and promote vascularization in rats with cerebral ischemia-reperfusion injury.