p53/miR-502-5p/TRAF2通路对骨关节炎软骨细胞损伤的影响

Effects of p53/miR-502-5p/TRAF2 pathway on chondrocyte damage in osteoarthritis

目的:探究微小RNA-502-5p(miR-502-5p)对白细胞介素-1β(IL-1β)诱导的骨关节炎(OA)软骨细胞损伤的影响。方法:Western blot和RT-q PCR检测骨关节炎患者软骨组织和IL-1β诱导的软骨细胞中p53和miR-502-5p的表达;分离培养软骨,并分组处理细胞,分别用MTT法检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测炎性因子和细胞外基质(ECM)相关蛋白的表达,另外,萤光素酶报告实验检测miR-502-5p对p53及肿瘤坏死因子受体相关因子2(TRAF2)的调控作用。结果:与正常软骨组织相比,OA患者软骨组织中miR-502-5p的水平显著降低,p53和TRAF2水平则明显升高(P<0.05)。IL-1β处理软骨细胞后,细胞活力下降、细胞凋亡率增加、IL-6、IL-8和TNF-α的蛋白表达水平显著上升;Ⅱ型胶原和蛋白聚糖的蛋白表达显著下调,基质金属蛋白酶(MMP)-3、MMP-9和MMP-13等的蛋白表达显著上调,miR-502-5p mimic转染反转了IL-1β对软骨细胞的这些作用。此外,p53与miR-502-5p之间存在负反馈调节,同时miR-502-5p能够靶向抑制TRAF2的水平。TRAF2沉默同样反转了IL-1β对软骨细胞增殖、凋亡、炎症反应以及ECM相关蛋白表达的影响。结论:调节p53/miR-502-5p/TRAF2通路能够减轻IL-1β诱导的骨关节软骨细胞损伤。

AIM： To explore the effects of microRNA-502-5 p（ miR-502-5 p） on interleukin-1β（ IL-1β）-induced chondrocyte damage. METHODS： The expression of p53 and miR-502-5 p in the cartilaginous tissues of the patients with osteoarthritis（ OA） and IL-1β-induced chondrocytes was detected by Western blot and RT-q PCR. The chondrocytes were isolated from normal cartilage tissues and given different treatments. The cell viability was measured by MTT assay.The cell apoptosis was analyzed by flow cytometry. The protein expression levels of proinflammatory-and extracellular matrix（ ECM）-related proteins were determined by Western blot. In addition,luciferase reporter assay was used to study the regulation between miR-502-5 p and p53/TRAF2. RESULTS： Compared with the normal cartilaginous tissues,the expression level of miR-502-5 p was decreased in the cartilaginous tissues of OA patients,while the expression levels of p53 and TRAF2 were significantly increased（ P〈0. 05）. After treatment with IL-1β,the viability of the chondrocytes was decreased,while the apoptotic rate and the protein levels of IL-6,IL-8 and TNF-α were increased. Moreover,the protein levels of collagen type Ⅱ and proteoglycans were decreased,while the protein levels of matrix metalloproteinase（ MMP）-3,MMP-9 and MMP-13 were increased obviously. However,transfection of miR-502-5 p mimic reversed the effects of IL-1βon the chondrocytes. Furthermore,miR-502-5 p-p53 negative feedback loop was found,miR-502-5 p inhibited TRAF2 by targeting TRAF2 gene. The silencing of TRAF2 also reversed the effects of IL-1β on the cell viability,apoptosis,inflammatory responses and ECM-related proteins expression in the chondrocytes. CONCLUSIONS： Regulation of p53/miR-502-5 p/TRAF2 pathway may decrease the IL-1β-induced chondrocyte damage.