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| 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 |
| 投稿时间：2018-12-03 修订日期：2019-03-06 |
| DOI： |
| 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) |
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| 中文摘要: |
| 目的：探究长链非编码RNA MEG3靶向miR-21的作用对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、MMP-13、Collagen II、Aggrecan基因表达水平,Hoechst检测细胞凋亡,Westernblot检测cl-Caspase-3、cl-Caspase-9、MMP-13、Collagen II、Aggrecan蛋白表达水平和p65、STAT3磷酸化比率,试剂盒检测MDA、LDH、SOD、GSH、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表达水平、细胞凋亡率、cl-caspase-3、cl-caspase-9蛋白表达水平、MMP-13基因和蛋白表达水平、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表达水平、Collagen II、Aggrecan基因和蛋白表达水平、SOD、GSH、IL-10水平降低；与IL-1β组比较,LV-MEG3组miR-21表达水平、细胞凋亡率、cl-caspase-3、cl-caspase-9蛋白表达水平、MMP-13基因和蛋白表达水平、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miR-21表达水平、细胞凋亡率、cl-caspase-3、cl-caspase-9蛋白表达水平、MMP-13基因和蛋白表达水平、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表达水平、Collagen II、Aggrecan基因和蛋白表达水平、SOD、GSH、IL-10水平降低；与miR-21 mimic组比较,LV-MEG3+mimic组miR-21表达水平、细胞凋亡率、cl-caspase-3、cl-caspase-9蛋白表达水平、MMP-13基因和蛋白表达水平、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。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 effects of long non-coding RNA MEG3 on IL-1β induced cell apoptosis , inflammatory response of chondrocytes by targeting miR-21 and its mechanism. Methods: Cells were divided into cTRL, IL-1β, LV-MEG3, miR-21 mimic and LV-MEG3+mimic groups, after cells were stimulated with IL-1β to mimick OA chondrocytes, each group were treated with corresponding miRNA or lentivirus. RT-PCR was performed for measuring the gene levels of MEG3, miR-21, MMP-13, Collagen II and Aggrecan, cell apoptosis was determined by Hoechst, Western blot was used to determine the protein levels of cl-Caspase-3, cl-Caspase-9, MMP-13, Collagen II, Aggrecan and phosphorylated ratios of p65, STAT3, MDA, LDH, SOD, GSH, TNF-α, IL-6, IL-110 levels were detected by kit, the nuclear localization signal of p65 was determined by immunofluorescence. Results: Compared with cTRL group, miR-21 level, cell apoptosis rate, cl-caspase-3 and cl-caspase-9 protein levels, MMP-13 gene and protein level, MDA、LDH、TNF-α、IL-6 levels, phosphorylated ratio of p65 and STAT3, nuclear localization of p65 were increased, and MEG3 level, gene and protein levels of Collagen II, Aggrecan, SOD, GSH, IL-10 levels were decreased in IL-1β group; Compared with IL-1β group, miR-21 level, cell apoptosis rate, cl-caspase-3 and cl-caspase-9 protein levels, MMP-13 gene and protein level, MDA、LDH、TNF-α、IL-6 levels, phosphorylated ratio of p65 and STAT3, nuclear localization of p65 were decreased, and MEG3 level, gene and protein levels of Collagen II, Aggrecan, SOD, GSH, IL-10 levels were increased in LV-MEG3 group, meanwhile, miR-21 level, cell apoptosis rate, cl-caspase-3 and cl-caspase-9 protein levels, MMP-13 gene and protein level, MDA、LDH、TNF-α、IL-6 levels, phosphorylated ratio of p65 and STAT3, nuclear localization of p65 were increased, and MEG3 level, gene and protein levels of Collagen II, Aggrecan, SOD, GSH, IL-10 levels were decreased in miR-21 mimic group; Compared with miR-21 mimic group, miR-21 level, cell apoptosis rate, cl-caspase-3 and cl-caspase-9 protein levels, MMP-13 gene and protein level, MDA、LDH、TNF-α、IL-6 levels, phosphorylated ratio of p65 and STAT3, nuclear localization of p65 were decreased, and MEG3 level, gene and protein levels of Collagen II, Aggrecan, SOD, GSH, IL-10 levels were increased in LV-MEG3+mimic group. Conclusion: MEG3 can inhibit the IL-1β induced cell apoptosis and alleviate the inflammatory response of chondrocytes by downregulate the expression of miR-21, its mechanism may related to inhibit NF-κB signal pathway. |
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