摘要
目的探讨微小RNA(miR)-214-3p在骨关节炎(OA)软骨细胞中的表达及其调节内皮细胞迁移和血管生成的机制。方法选取2020年6月至2020年12月在郑州大学第一附属医院接受全膝关节置换的16例OA患者及12例无膝关节疾病但因创伤导致下肢截肢的患者分别作为OA组和健康组,获取软骨标本,提取软骨细胞。采用蛋白印迹分析和实时定量反转录聚合酶链反应(RT-qPCR)检测miR-214-3p在软骨细胞中的表达水平。通过创面愈合实验、小管形成实验、酶联免疫吸附法(ELISA)检测细胞的迁移率、血管生成率、血管内皮生长因子(VEGF)分泌情况。采用生物信息学分析、双荧光素酶分析等方法分析miR-214-3p与神经营养酪氨酸激酶受体2型(TrkB)的关系。采用方差分析(ANOVA)和t检验。结果TrkB在OA组中的表达水平明显高于健康组(2.13±0.41比0.92±0.21,t=9.318,P<0.01)。miR-214-3p在OA组中的表达明显低于健康组(0.26±0.12比1.12±0.31,t=-10.177,P<0.01),差异均有统计学意义。OA组miR-214-3p表达水平与TrkB表达水平呈负相关(r=-0.730,P<0.05),健康组中miR-214-3p表达水平与TrkB表达水平呈负相关(r=-0.705,P<0.05)。TargetScan数据库预测了miR-214-3p和TrkB的靶向结合位点,双荧光素酶实验验证了TrkB与miR-214-3p的靶向结合。TrkB过表达组VEGF水平高于空载体组[(221.3±5.2)ng/L比(137.2±4.8)ng/L,t=43.742,P<0.01]、内皮细胞迁移率高于空载体组(1.71±0.22比0.98±0.11,t=10.516,P<0.05)、血管生成率高于空载体组(1.41±0.12比0.99±0.10,t=9.822,P<0.05),差异均有统计学意义。下调OA组的miR-214-3p表达后,VEGF表达水平升高0.95倍(t=5.245,P<0.01),内皮细胞迁移率增加1.12倍(t=3.283,P<0.01),血管生成率增加0.52倍(t=1.174,P<0.01),差异均有统计学意义。结论软骨细胞中TrkB信号通路旁分泌VEGF的激活是由miR-214-3p介导的,在OA发展过程中调控内皮细胞迁移和血管生成。
Objective To study the expression of microRNA(miR)-214-3p in osteoarthritis(OA)chondrocytes and its mechanism of regulating endothelial cell migration and angiogenesis.Methods A total of 16 patients with OA who underwent total knee arthroplasty in the First Affiliated Hospital of Zhengzhou University from June 2020 to December 2020 and 12 patients without knee disease but lower limb amputation due to trauma were selected as OA group and healthy group respectively.Cartilage samples were obtained and chondrocytes were extracted.Western blotting and real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)were used to detect the expression level of miR-214-3p in chondrocytes.The cell migration rate,angiogenesis rate and the secretion of vascular endothelial growth factor(VEGF)were detected by wound healing experiment,tubule formation experiment and enzyme-linked immunosorbent assay(ELISA).The relationship between miR-214-3p and neurotrophic tyrosine kinase receptor type 2(TrkB)was analyzed by bioinformatics analysis and double luciferase analysis.The data were analyzed by statistical software,and the statistical differences were determined by analysis of variance(ANOVA)and t-test.Results The expression level of TrkB in OA group was significantly higher than that in healthy group(2.13±0.41 vs.0.92±0.21,t=9.318,P<0.01).The expression of miR-214-3p in OA group was significantly lower than that in healthy group(0.26±0.12 vs.1.12±0.31,t=-10.177,P<0.01).The expression level of miR-214-3p in OA group was negatively correlated with the expression level of TrkB(r=-0.730,P<0.05),and the expression level of miR-214-3p in healthy group was negatively correlated with the expression level of TrkB(r=-0.705,P<0.05).Targetscan database predicted the targeted binding sites of miR-214-3p and TrkB.Double luciferase experiment verified the targeted binding of TrkB to miR-214-3p.The VEGF level in TrkB overexpression group was higher[(221.3±5.2)ng/L vs.(137.2±4.8)ng/L,t=43.742,P<0.01],the endothelial cell migration rate was higher(1.71±0.22 vs.0.98±0.11,t=10.516,P<0.05),and the angiogenesis rate was higher(1.41±0.12 vs.0.99±0.10,t=9.822,P<0.05)than in empty body group.After down-regulating the expression of miR-214-3p in OA group,the expression level of VEGF increased by 0.95 times(t=5.245,P<0.01),the migration rate of endothelial cells increased by 1.12 times(t=3.283,P<0.01),and the angiogenesis rate increased by 0.52 times(t=1.174,P<0.01).Conclusion The activation of paracrine VEGF in TrkB signaling pathway is mediated by miR-214-3p,which regulates endothelial cell migration and angiogenesis during the development of OA.
作者
任泰超
肖鹏
Ren Taichao;Xiao Peng(Department of Orthopeadics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华实验外科杂志》
CAS
北大核心
2022年第1期116-119,共4页
Chinese Journal of Experimental Surgery
