miR-515-5p靶向Toll样受体4调控髓样分化因子88/NF-κB通路抑制骨关节炎软骨细胞凋亡及炎症反应的分子机制研究

miR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes

目的探究miR-515-5p抑制骨关节炎(osteoarthritis,OA)软骨细胞凋亡、缓解炎症反应的分子机制。方法体外培养人软骨细胞系C28/I2,使用10 ng/mL IL-1β处理细胞24 h构建体外OA模型;另外,分别采用miR mimics、mimics阴性对照(negative control,NC)、过表达(over expression,oe)-NC和oe-Toll样受体4(Toll-like receptor 4,TLR4)转染C28/I2细胞后,使用10 ng/mL IL-1β处理各组细胞24 h构建OA模型。采用细胞计数试剂盒8和EdU检测细胞增殖能力,流式细胞术检测细胞凋亡和细胞周期,Western blot检测B淋巴细胞瘤2蛋白(B-cell lymphoma 2 protion,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、裂解的半胱天冬酶3(cleaved-Caspase-3)、TLR4、髓样分化因子88(myeloid differentiation primary response gene 88,MyD88)、p65及磷酸化p65(phosphorylated p65,p-p65)蛋白的表达水平,实时荧光定量PCR检测miR-515-5p、TLR4mRNA表达水平,ELISA检测细胞上清液中促炎因子前列腺素E2(prostaglandin E2,PGE2)、TNF-α、IL-6的水平。通过BiBiServ2数据库预测miR-515-5p和TLR4之间的潜在结合位点,并采用双荧光素酶报告实验验证miR-515-5p和TLR4的靶向关系。结果采用IL-1β处理C28/I2细胞后,miR-515-5p、Bcl-2蛋白的表达及细胞增殖能力均显著降低,Bax和cleaved-Caspase-3蛋白表达水平、细胞上清液中促炎因子(PGE2、TNF-α、IL-6)水平及细胞凋亡率均显著增加;此外,S期和G2期细胞比例显著降低,G1期细胞比例显著增加,提示IL-1β处理后细胞周期受到阻滞。而转染miR mimics后,细胞中miR-515-5p表达水平显著上调,部分逆转了IL-1β诱导的OA软骨细胞凋亡,缓解了OA软骨细胞的周期阻滞和炎症反应。采用IL-1β处理C28/I2细胞后,TLR4的mRNA和蛋白水平均显著升高;过表达miR-515-5p后,靶向抑制了TLR4的表达并且阻断了MyD88/NF-κB通路的激活。而过表达TLR4可部分逆转miR mimics对IL-1β诱导的OA软骨细胞凋亡及炎症的改善作用。结论miR-515-5p靶向负调控TLR4的表达,抑制了MyD88/NF-κB通路激活以及OA软骨细胞凋亡,并有效缓解了细胞炎症反应。

Objective To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis(OA).Methods Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β(IL-1β)for 24 hours to construct an in vitro OA model.C28/I2 cells were transfected with miR mimics,mimics negative control(NC),over expression(oe)-NC,and oe-Toll-like receptor 4(TLR4),respectively,and then treated with 10 ng/mL IL-1βfor 24 hours to establish OA model.Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2’-deoxyuridine,cell apoptosis and cell cycle were detected by flow cytometry,and B-cell lymphoma 2 protion(Bcl-2),Bcl-2-associated X protein(Bax),cleaved-Caspase-3,TLR4,myeloid differentiation primary response gene 88(MyD88),p65 and phosphorylated p65(p-p65)protein expression levels were detected by Western blot.Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4,and ELISA was used to detect pro-inflammatory factor prostaglandin E2(PGE2),tumor necrosis factorα(TNF-α),and IL-6 levels in cell supernatant.The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database,and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay.Results After the treatment of C28/I2 cells with IL-1β,the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced.The expression levels of Bax and cleaved-Caspase-3 protein,the levels of pro-inflammatory factors(PGE2,TNF-α,IL-6)in the supernatant of C28/I2 cells,and the apoptosis of C28/I2 cells significantly increased.In addition,the proportion of the cells at S phase and G2 phase decreased significantly,and the proportion of cells at G1 phase increased significantly,suggesting that the cell cycle was blocked after IL-1βtreatment.After transfection with miR mimics,the expression level of miR-515-5p in the cells significantly upregulated,partially reversing the apoptosis of OA chondrocytes induced by IL-1β,and alleviating the cycle arrest and inflammatory response of OA chondrocytes.After treating C28/I2 cells with IL-1β,the mRNA and protein levels of TLR4 significantly increased.Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factorκB(NF-κB)pathway.Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes.Conclusion miR-515-5p negatively regulates the expression of TLR4,inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes,and effectively alleviates the inflammatory response of the cells.