基质细胞衍生因子1诱导骨关节炎软骨细胞的miRNA表达谱分析

MicroRNA expression profiles of chondrocytes in osteoarthritis induced by stromal cell derived factor 1

背景:骨关节炎是多因素介导的复杂疾病,发病机制尚待挖掘,随着基因层面的不断深入研究,非编码核糖核酸调控作用显现并得以研究。通过检测软骨细胞退变miRNA表达谱变化有助于更好地理解骨软骨细胞退变的分子机制,并为骨关节炎的诊断和治疗开辟新的途径。目的:探讨基质细胞衍生因子1刺激骨关节炎软骨细胞后miRNA表达谱变化,为基因层面延缓关节软骨退变提供实验基础。方法:对10例膝关节骨关节炎患者于全膝关节置换手术过程中截骨后残留的软骨组织进行软骨细胞培养,随机分为实验组与对照组,两组细胞培养基为含体积分数10%胎牛血清及青链霉素双抗的高糖DMEM培养基。实验组培养基中另外加入100μg/L基质细胞衍生因子1,对照组不做任何处理。两组软骨细胞培养48 h后,进行下一步实验用于miRNA芯片筛选和实时定量PCR验证。软骨组织标本取材前皆告知患者并征得同意,该研究符合《医疗机构管理条例》相关要求,获得昆明医科大学第一附属医院伦理委员会批准。结果与结论:miRNA基因芯片初筛共有84个miRNAs发生变化,其中70个miRNAs上调,14个miRNAs下调。通过基因芯片筛选差异变化的miRNA,对变化显著的7个miRNA(miR-146a-5p、miR-124-3p、miR-130a-3p、miR-185-5p、miR-221-3p、miR-126-3p)进行qRT-PCR实验验证,其中miR-146a-5p、miR-124-3p及miR-126-3p的qRT-PCR结果与基因芯片结果一致。结果表明,基质细胞衍生因子1刺激骨关节炎软骨细胞后循环miRNA表达谱出现明显变化,miR-146a-5p、miR-124-3p及miR-126-3p可能与基质细胞衍生因子1刺激骨关节炎SDF-1/CXCR4信号通路反应有关。

BACKGROUND:Osteoarthritis is a complex disease caused by many factors,but its pathogenesis is yet unknown.Intensive molecular genetic research has indicated that noncoding RNA may be a potential transcriptional regulatory factor in osteoarthritis development.Exploration on differentially expressed miRNAs between osteoarthritis and normal tissue gives the clue to understand the molecular mechanism of osteoarthritis,providing a new cue for the diagnosis and treatment of osteoarthritis.OBJECTIVE:To discuss the changes of miRNA expression profile of chondrocytes stimulated by stromal cell derived factor 1 in osteoarthritis,and provide an experimental basis for delaying articular cartilage degeneration at the genetic level.METHODS:Residual cartilage samples from 10 patients with knee osteoarthritis who underwent total knee replacement were collected for culture of chondrocytes.The cells were then randomized into two groups:experimental group was cultured in high-glucose DMEM medium containing 10%fetal bovine serum and penicillin with the addition of 100μg/L stromal cell derived factor 1,and control group was cultured in the medium with no other induction.At 48 hours after cell culture,the miRNA expression profiles of chondrocytes were detected,and the results were verified by fluorescence quantitative RT-PCR.The study protocol was performed in accordance with the Regulations on the Administration of Medical Institutions.All study patients gave written informed consent before collection of cartilage tissue.RESULTS AND CONCLUSION:After preliminary screening,84 microRNAs were altered,of which 70 were up-regulated and 14 were down-regulated.Seven microRNAs with significant changes(miR-146a-5p,miR-124-3p,miR-130a-3p,miR-185-5p,miR-221-3p,miR-126-3p)were selected by gene chip for qRT-PPCR experiment,which indicated that the qRT-PCR results of miR-146a-5p,miR-124-3p and miR-126-3p were consistent with the results of gene chip.Therefore,induction with stromal cell derived factor 1 makes the expression profile of circulating miRNA change a lot.miR-146a-5p,miR-124-3p and miR-126-3p may be related to the stromal cell derived factor 1/CXCR4 signaling pathway by which stromal cell derived factor 1 can stimulate osteoarthritis.