摘要
背景:滑膜炎症参与骨关节炎的所有阶段,是促进骨关节炎发生发展的关键因素。研究表明,环状RNA(circRNA)在滑膜细胞及软骨细胞增殖、凋亡和细胞外基质降解过程中发挥着重要作用。目的:观察circRNA SEC24A对白细胞介素1β诱导的人滑膜成纤维细胞增殖、凋亡及炎症因子表达的影响。方法:将人滑膜成纤维细胞分为4组,包括对照组、白细胞介素1β组、空载体组、sh-circSEC24A组。除对照组外,其余3组用10 ng/mL白细胞介素1β诱导24 h建立炎症细胞模型;空载体组、sh-circSEC24A组感染空载体病毒和敲低circSEC24A的慢病毒载体。CCK-8法检测细胞增殖情况;流式细胞术检测细胞凋亡情况;ELISA法检测细胞上清液中基质金属蛋白酶9、基质金属蛋白酶13、白细胞介素6和肿瘤坏死因子α水平;Western blot法检测细胞中Bax、Bcl-2、基质金属蛋白酶9、基质金属蛋白酶13、casepase3、cleaved-casepase3、casepase8和cleavedcasepase8蛋白相对表达量。结果与结论:(1)qRT-PCR结果显示,与正常组相比,白细胞介素1β诱导的人滑膜成纤维细胞中circSEC24A表达显著上调;(2)CCK-8法检测sh-circSEC24A组细胞吸光度值显著高于白细胞介素1β组和空载体组(P<0.05),流式细胞术检测sh-circSEC24A组细胞凋亡率显著低于白细胞介素1β组和空载体组(P<0.05);(3)ELISA法检测sh-circSEC24A组人滑膜成纤维细胞上清液中肿瘤坏死因子α和白细胞介素6水平显著低于白细胞介素1β组和空载体组(P<0.01,P<0.001);(4)Western blot结果显示,与白细胞介素1β组和空载体组相比,sh-circSEC24A组促凋亡因子Bax蛋白表达显著下降,抑凋亡因子Bcl-2蛋白表达显著上升(P<0.05);凋亡以及相关活化因子cleaved-casepase3和cleaved-casepase8蛋白表达均降低(P<0.05);(5)ELISA和Western blot结果显示,与白细胞介素1β组和空载体组相比,sh-circSEC24A组基质金属蛋白酶9、基质金属蛋白酶13蛋白表达显著降低(P<0.05)。结果表明,白细胞介素1β诱导的人滑膜成纤维细胞中circSEC24A表达异常增高,敲低circSEC24A表达可促进人滑膜成纤维细胞增殖,抑制细胞凋亡、炎症因子释放和细胞外基质降解,提示circSEC24A可能是早期骨关节炎的重要干预靶点。
BACKGROUND:Synovitis is involved in all stages of osteoarthritis and is a key factor contributing to the development of osteoarthritis.Studies have shown that circular RNA(circRNA)plays an important role in the proliferation,apoptosis and extracellular matrix degradation of synovial cells and chondrocytes.OBJECTIVE:To observe the effects of circRNA SEC24A on the interleukin-1β-induced proliferation,apoptosis,and expression of inflammatory factors in human synovial fibroblasts.METHODS:Human synovial fibroblasts were divided into four groups,including control group,interleukin-1βgroup,empty vector group,and sh-circSEC24A group.Except for the control group,the other three groups were induced with 10 ng/mL interleukin-1βfor 24 hours to establish inflammatory cell models;the empty vector group and sh-circSEC24A group were infected with empty vector virus and lentiviral vector knocking down circSEC24A.CCK-8 assay was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.ELISA was used to detect the levels of matrix metalloproteinase-9,matrix metalloproteinase-13,interleukin-6,and tumor necrosis factor-αin cell supernatant.Western blot assay was used to detect the relative expression levels of Bax,Bcl-2,matrix metalloproteinase-9,matrix metalloproteinase-13,casepase3,cleaved-casepase3,casepase8,and cleaved-casepase8 proteins in cells.RESULTS AND CONCLUSION:(1)qRT-PCR results showed that compared with the normal group,the expression of circSEC24A in human synovial fibroblasts induced by interleukin 1βwas significantly up-regulated.(2)The absorbance value of cells in the sh-circSEC24A group detected by CCK-8 assay was significantly higher than that of interleukin 1βgroup and empty vector group(P<0.05).The apoptosis rate of sh-circSEC24A group detected by flow cytometry was significantly lower than that of interleukin 1βgroup and empty vector group(P<0.05).(3)The levels of tumor necrosis factorαand interleukin 6 in the supernatant of human synovial fibroblasts in the sh-circSEC24A group detected by ELISA were significantly lower than those in the interleukin 1βgroup and the empty vector group(P<0.01,P<0.001).(4)Western blot assay results showed that compared with the interleukin 1βgroup and the empty vector group,the expression of the pro-apoptotic factor Bax protein in the sh-circSEC24A group significantly decreased,and the expression of the anti-apoptotic factor Bcl-2 protein increased significantly(P<0.05);apoptosis and related activating factors cleaved-casepase3 and cleaved-casepase8 protein expressions were both reduced(P<0.05).(5)ELISA and western blot assay results showed that compared with the interleukin 1βgroup and the empty vector group,the sh-circSEC24A group had lower levels of matrix metalloproteinase 9 and matrix metalloproteinase 13 protein(P<0.05).These findings indicated that the expression of circSEC24A was abnormally increased in human synovial fibroblasts induced by interleukin 1β.Knocking down circSEC24A expression could promote the proliferation of human synovial fibroblasts and inhibit apoptosis,inflammatory factor release,and extracellular matrix degradation,suggesting that circSEC24A may be an important intervention target for early osteoarthritis.
作者
周丽君
张克远
徐飞虎
王茜
俞丽
董士铭
徐俊宇
郭宇沨
马海蓉
丁红
Zhou Lijun;Zhang Keyuan;Xu Feihu;Wang Xi;Yu Li;Dong Shiming;Xu Junyu;Guo Yufeng;Ma Hairong;Ding Hong(School of Public Health,Xinjiang Medical University,Urumqi 830011,Xinjiang Uygur Autonomous Region,China;Institute of Clinical Medicine,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang Uygur Autonomous Region,China;Karamay Central Hospital,Karamay 834000,Xinjiang Uygur Autonomous Region,China;Department of Spine and Orthopedics,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang Uygur Autonomous Region,China)
出处
《中国组织工程研究》
北大核心
2025年第24期5086-5092,共7页
Chinese Journal of Tissue Engineering Research
基金
新疆维吾尔自治区自然科学基金项目(2024D01C136),项目负责人:丁红
新疆维吾尔自治区“十四五”高等学校特色学科-公共卫生与预防医学
新疆维吾尔自治区自然科学基金重点项目(2021D01D21),项目负责人:马海蓉。
