中国骨质疏松杂志

长链非编码RNA MEG3靶向下调miR-21对IL-1β诱导的软骨细胞凋亡及炎症反应的影响

The effects of long non-coding RNA MEG3 on IL-1β-induced cell apoptosis and inflammatory response of chondrocytes by targeting miR-21

DOI：10.3969/j.issn.1006.7108.2020.04.010

中文关键词: 骨关节炎软骨细胞 MEG3 miR-21

英文关键词:osteoarthritis chondrocyte MEG3 miR-21

基金项目:河南省卫生厅重点基金资助项目(2359101)

作者	单位
丁童* 周彦鹏冯立平	新乡医学院附属中心医院创伤外科，河南新乡453003

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中文摘要:

目的探究长链非编码RNA母系表达基因3（MEG3）靶向miR-21的作用对白细胞介素1β（IL-1β）诱导的软骨细胞凋亡及炎症反应的影响及其作用机制。方法将细胞分为cTRL组、IL-1β组、LV-MEG3组、miR-21 mimic组和LV-MEG3+mimic组，用IL-1β处理软骨细胞后，加入对应的慢病毒或miRNA mimic处理细胞。RT-PCR检测MEG3、 miR-21、基质金属蛋白酶13（MMP-13）、II型胶原蛋白（Collagen II）、聚蛋白聚糖（Aggrecan）基因表达水平，Hoechst检测细胞凋亡，Western blot检测活化半胱天冬酶3（cl-Caspase-3）、cl-Caspase-9、MMP-13、Collagen II、Aggrecan蛋白表达水平和p65、信号传导及转录激活因子3（STAT3）磷酸化比率，试剂盒检测丙二醛（MDA）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、肿瘤坏死因子α（TNF-α）、IL-6、IL-10水平，免疫荧光检测p65的核定位情况。结果与cTRL组比较，IL-1β组miR-21表达，细胞凋亡率，MMP-13基因表达，cl-Caspase-3、cl-Caspase-9、MMP-13蛋白表达，MDA、LDH、TNF-α、IL-6水平，p65和STAT3磷酸化比率，p65核内信号水平升高，MEG3表达，Collagen II、Aggrecan基因和蛋白表达，SOD、GSH、IL-10水平降低；与IL-1β组比较，LV-MEG3组miR-21表达，细胞凋亡率，MMP-13基因表达，cl-Caspase-3、cl-Caspase-9、MMP-13蛋白表达，MDA、LDH、TNF-α、IL-6水平，p65和STAT3磷酸化比率，p65核内信号水平降低，MEG3表达水平，Collagen II、Aggrecan基因和蛋白表达水平，SOD、GSH、IL-10水平升高；miR-21 mimic组各项检测指标的变化与LV-MEG3组相反；与miR-21 mimic组比较，LV-MEG3+mimic组miR-21表达，细胞凋亡率，MMP-13基因表达，MMP-13、cl-Caspase-3、cl-Caspase-9蛋白表达，MDA、LDH、TNF-α、IL-6水平，p65和STAT3磷酸化比率，p65核内信号水平降低，MEG3表达水平，Collagen II、Aggrecan基因和蛋白表达，SOD、GSH、IL-10水平升高。结论 MEG3可下调miR-21表达，从而抑制IL-1β诱导的软骨细胞凋亡，缓解炎症反应，其作用机制可能与抑制NF-κB信号通路激活有关。

英文摘要:

Objective To investigate the effect of long non-coding RNA MEG3 on IL-1β-induced cell apoptosis and inflammatory response of chondrocytes by targeting miR-21 and its mechanism. Methods Cells were divided into cTRL group, IL-1β group, LV-MEG3 group, miR-21 mimic group, and LV-MEG3+mimic group. After addition of IL-1β, cells were treated with corresponding miRNA or lentivirus. Gene levels of MEG3, miR-21, MMP-13, collagen II, and Aggrecan were measured with RT-PCR. Cell apoptosis was determined with Hoechst staining. The protein levels of cl-Caspase-3, cl-Caspase-9, MMP-13, Collagen II, Aggrecan, phosphorylated ratios of p65, and STAT3 were determined with Western blotting. The levels of MDA, LDH, SOD, GSH, TNF-α, IL-6, and IL-10 were determined using the detecting kits. The nuclear localization of p65 was determined with immunofluorescence. Results Compared with those in cTRL group, miR-21 level, cell apoptosis rate, MMP-13 gene level, MMP-13, cl-caspase-3 and cl-caspase-9 protein levels, MDA, LDH, TNF-α, IL-6 levels, phosphorylated ratio of p65 and STAT3, nuclear localization of p65 increased, but MEG3 level, gene and protein levels of collagen II and Aggrecan, SOD, GSH, IL-10 levels decreased in IL-1β group. Compared with those in IL-1β group, miR-21 level, cell apoptosis rate, MMP-13 gene level, MMP-13, cl-caspase-3 and cl-caspase-9 protein levels, MDA, LDH, TNF-α, IL-6 levels, phosphorylated ratio of p65 and STAT3, nuclear localization of p65 decreased, but MEG3 level, gene and protein levels of collagen II and Aggrecan, SOD, GSH, IL-10 levels increased in LV-MEG3 group. Meanwhile, the alterations in miR-211 mimic group were opposite to those in LV-MEG3 group. Compared with those in miR-21 mimic group, miR-21 level, cell apoptosis rate, MMP-13 gene level, MMP-13, cl-caspase-3 and cl-caspase-9 protein levels, MDA, LDH, TNF-α, IL-6 levels, phosphorylated ratio of p65 and STAT3, nuclear localization of p65 decreased, but MEG3 level, gene and protein levels of collagen II, Aggrecan, SOD, GSH, IL-10 levels increased in LV-MEG3+mimic group. Conclusion MEG3 inhibits IL-1β induced cell apoptosis and alleviates the inflammatory response of chondrocytes by down-regulation of the expression of miR-21, and its mechanism may be related to the inhibition of NF-κB signal pathway.

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