Effect and mechanism of miR-148a-3p on chondrocyte inflammation and apoptosis
Objective To study the effects of miR-148a-3p on inflammatory cytokines,apoptosis,and phenotype of chondrocytes.Methods Primary chondrocytes were extracted from 2-week-old large-eared rabbits after euthanasia.To explore the effects of inflamma-tion on cartilage-related markers and intracellular miR-148a-3p expression levels,chondrocytes were divided into control group and IL-1 β group.To explore the effects of miR-148a-3p on the expression levels of cartilage-related and apoptosis-related markers,chondro-cytes were divided into blank group,IL-1 β group,IL-1 β+NC group,IL-1 β+mimics group,and IL-1 β+inhibitor group.qRT-PCR was used to measure IL-1 β,SOX9,COL-Ⅱ,ACAN,MMP-13 mRNA levels.Western blot was used to measure the protein expressions of cartilage-related markers MMP-13,COL-Ⅱ,ACAN,and SOX9,and cell apoptosis-related markers Bax,Caspase-3,and Bcl-2.ELISA was used to measure IL-6 and IL-10 levels in cell supernatants.Results ①Compared with control group,MMP-13 expression was increased in IL-1 β group(P＜0.05),and COL-Ⅱ and ACAN expression levels were decreased(P＜0.05).②Compared with blank group,IL-6 level was increased while IL-10 level was decreased in cell supernatants in IL-1 β group(P＜0.05),and the mRNA and protein expression levels of MMP-13 were increased while the mRNA and protein expression levels of COL-Ⅱ and ACAN were decreased(P＜0.05).Compared with IL-1 β group,the level of IL-6 in cell supernuents was increased in IL-1 β+mimics group(P＜0.05),MMP-13 mRNA and protein expression levels,and Bax and Caspase-3 protein expressions were increased(P＜0.05),while the level of IL-10 in cell supernuents,SOX9,COL-Ⅱ,ACAN mRNA and protein levels,and Bcl-2 protein expression levels were decreased(P＜0.05).There was no statistically significant difference of the above indexes in IL-1 β+inhibitor group and IL-1 β group(P＞0.05).Conclusion IL-1 β can induce the upregulation of miR-148a-3p expression in chondrocytes in vitro,which leads to the inflammatory changes,apoptosis,phenotypic changes of chondrocytes,and extracellular matrix(ECM)degradation.
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