目的 探讨长链非编码 RNA MIR155 宿主基因(MIR155 host gene,MIR155HG)对白介素-1β(interleukin-1β,IL-1β)诱导的骨关节炎软骨细胞炎性损伤的调控作用和机制.方法 采用 10 ng/mL的IL-1β刺激软骨细胞 24h来建立骨关节炎的细胞模型.软骨细胞分为对照组、模型组(IL-1β刺激软骨细胞)、模型+si-阴性对照(negative control,NC)组(转染NC siRNA 48 h,再进行IL-1β刺激)和模型+ si-MIR155HG组(转染MIR155HG siRNA 48 h,再进行IL-1β刺激).采用实时荧光定量逆转录聚合酶链反应RT-qPCR检测MIR155HG的表达水平.采用钙黄绿素乙酰氧基甲酯(Calcein acetoxymethyl ester,Calcein AM)细胞活性检测试剂盒检测软骨细胞的存活率.采用原位末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)检测软骨细胞凋亡.采用Western blot检测B细胞淋巴瘤/白血病-2(B-cell CLL/lymphoma-2,Bcl-2)、Bcl-2 相关 X 蛋白(Bcl-2-associated X protein,Bax)、基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)、Ⅱ型胶原α1(collagen type Ⅱ alpha 1,COL2A1)、含NLR家族 Pyrin域蛋白 3(NLR family pyrin domain containing protein 3,NLRP3)、凋亡相关斑点样蛋白(apoptosis associated speck like protein containing a CARD,ASC)和 Caspase-1 的蛋白表达水平的蛋白表达水平.采用酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)试剂盒检测细胞炎症因子白介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白介素-18(interleukin-18,IL-18)的浓度.结果 与对照组比较,模型组的MIR155HG表达水平显著升高(P<0.01).与模型+ si-NC组比较,模型+ si-MIR155HG组的MIR155HG表达水平显著降低(P<0.01).与对照组比较,模型组和模型+ si-NC组软骨细胞的存活率显著降低(P<0.01),细胞凋亡水平显著升高(P<0.01),细胞外基质降解显著增加(P<0.01),IL-6、TNF-α和IL-18 的浓度显著升高(P<0.01),NLRP3、ASC和Caspase-1 的蛋白表达水平显著升高(P<0.01).与模型组和模型+ si-NC组比较,模型+ si-MIR155HG组软骨细胞的存活率显著提高(P<0.01),细胞凋亡水平显著下降(P<0.01),细胞外基质降解显著降低(P<0.01),IL-6、TNF-α和IL-18 浓度显著下降(P<0.01),NLRP3、ASC和Caspase-1的蛋白表达水平显著降低(P<0.01).结论 基因沉默lncRNA MIR155HG可以缓解IL-1β诱导的软骨细胞炎症损伤,其保护作用可能是通过抑制NLRP3 炎症小体的激活来实现的.
Long non-coding RNA MIR155HG regulates IL-1β-induced inflammatory injury in osteoarthritis chondrocytes
Objective To explore the regulatory effect of long non-coding RNA MIR155 host gene(MIR155HG)on interleukin 1β(IL-1β)-induced inflammatory injury of osteoarthritis chondrocytes and the underlying mechanism.Methods The in vitro model of osteoarthritis was established by stimulating chondrocytes with IL-1β at 10ng/mL for 24h.The chondrocytes were treated with blank control,induced with IL-1β,transfected with NC siRNA(si-NC)for 48h and induced with IL-1β,or transfected with MIR155HG siRNA(si-MIR155HG)for 48 h and induced with IL-1β.The expression level of MIR155HG was examined using the real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR).The survival rate of chondrocytes was detected using the Calcein AM Cell Viability Assay Kit.The apoptosis of chondrocytes was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)Apoptosis Assay Kit.The protein levels of Bax,Bcl-2,matrix metallopeptidase-13(MMP-13),collagen type Ⅱ alpha 1(COL2A1),NLR-family pyrin domain-containing protein 3(NLRP3),ASC,and Caspase-1 were measured by Western blot.The concentrations of interleukin 6(IL-6),tumor necrosis factor-alpha(TNF-α),and interleukin 18(IL-18)were quantified by the enzyme linked immunosorbent assay(ELISA)kits.Results Compared with that of blank control,MIR155HG was significantly upregulated in IL-1β-induced chondrocytes(P<0.01).It was significantly downregulated in IL-1β-induced chondrocytes transfected with si-MIR155HG than those transfected with si-NC(P<0.01).Compared with that of blank control,IL-1β-induced chondrocytes and those transfected with si-NC presented significantly lower cell viability,and significantly higher apoptotic rate,degradation of extracellular matrix,relative levels of IL-6,TNF-α and IL-18 and protein levels of NLRP3,ASC and caspase-1(all P<0.01).Compared with those induced with IL-1β with either the transfection of si-NC or not,IL-1β-induced chondrocytes transfected with si-MIR155HG presented significantly higher cell viability,and significantly lower apoptotic rate,degradation of extracellular matrix,relative levels of IL-6,TNF-α and IL-18 and protein levels of NLRP3,ASC and caspase-1(all P<0.01).Conclusion Silence of lncRNA MIR155HG ameliorates IL-1β-induced inflammatory injury of chondrocytes by inhibiting the activation of NLRP3 inflammasome.
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维普
