lncRNA GPRC5D-AS1对地塞米松诱导小鼠成肌细胞肌萎缩的抵抗和再生作用及其机制  

Resistance and regeneration effects of lncRNA GPRC5D-AS1 on muscle atrophy of myocytes in mice induced by dexamethasone and its mechanism

作　　者：张瑞鹏 李杰[1] ZHANG Ruipeng;LI Jie(Cadre Ward,First Hospital,Jilin University,Changchun 130021,China)

机构地区：[1]吉林大学第一医院干部病房,吉林长春130021

出　　处：《吉林大学学报（医学版）》2023年第6期1457-1465,共9页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅自然科学基金项目(20200201118JC);吉林省发改委自主创新能力建设项目(2020C052)。

摘　　要：目的:探讨长链非编码RNA(lncRNA)GPRC5D-AS1对地塞米松诱导的小鼠成肌细胞肌萎缩的抵抗和再生作用,并阐明其作用机制。方法:选取对数生长期C2C12细胞,检测0、25、50、75、100、200和400 mg·L^(-1)地塞米松诱导24、48和72 h后C2C12细胞存活率,筛选肌萎缩模型诱导的最佳作用剂量和时间。实时荧光定量PCR(RT-qPCR)法检测C2C12细胞中lncRNA GPRC5D-AS1表达水平以验证细胞模型。将C2C12细胞分为正常组、模型组、lncRNA GRPC5DAS1-NC组和lncRNA GRPC5D-AS1-OE组,RT-qPCR法检测各组细胞中lncRNA GPRC5D-AS1表达水平,流式细胞术检测各组细胞中活性氧(ROS)水平,酶联免疫吸附试验(ELISA)法检测各组细胞中谷胱甘肽过氧化酶4(GPX4)、铁离子(Fe2+)和丙二醛(MDA)水平,透射电镜观察各组细胞线粒体超微结构表现,免疫荧光法检测各组细胞中成肌分化蛋白(MyoD)表达情况,Western blotting法检测各组细胞中铁死亡相关蛋白表达水平。结果:100 mg·L^(-1)地塞米松诱导48 h时造模效果最佳。与正常C2C12细胞比较,100 mg·L^(-1)地塞米松诱导48 h后C2C12细胞中lncRNA GPRC5DAS1表达水平降低(P<0.05),证实lncRNA GPRC5D-AS1在肌萎缩模型中有调控作用。RT-qPCR法检测,与正常组比较,模型组细胞中lncRNA GRPC5D-AS1表达水平降低(P<0.05);与模型组和lncRNA GRPC5D-AS1-NC组比较,lncRNA GRPC5D-AS1-OE组细胞中lncRNA GRPC5D-AS1表达水平升高(P<0.05)。流式细胞术检测,与正常组比较,模型组细胞中ROS水平升高(P<0.05);与模型组和lncRNA GRPC5D-AS1-NC组比较,lncRNA GRPC5D-AS1-OE组细胞中ROS水平降低(P<0.05)。ELISA法检测,与正常组比较,模型组细胞中Fe2+和MDA水平升高(P<0.05),GPX4水平降低(P<0.05);与模型组和lncRNA GRPC5D-AS1-NC组比较,lncRNA GRPC5D-AS1-OE组细胞中Fe2+和MDA水平降低(P<0.05),GPX4水平升高(P<0.05)。透射电镜观察,正常组细胞外形圆整,大小正常,膜上绒毛丰富,线粒体细胞器形态正常;模型组细胞中线粒�Objective:To discuss the resistance and regeneration effects of long non-coding RNA(lncRNA)GPRC5D-AS1 on the muscle atrophy of myocytes in the mice induced by dexamethasone,and to clarify its mechanism.Methods:The C2C12 cells at logarithmic growth phase were selected.The survival rates of the cells after treated with 0,25,50,75,100,200 and 400 mg·L^(-1) dexamethasone for 24,48 and 72 h were detected,which was used to select the optimal dose and time to induce the muscle atrophy model.The expression level of lncRNA GPRC5D-AS1 in the C2C12 cells was detected by real-time fluroscence quantitative PCR(RT-qPCR)method to validate the cell model.The C2C12 cells were divided into normal group,control group,lncRNA GPRC5D-AS1-NC group,and lncRNA GPRC5D-AS1-OE group.The expression levels of lncRNA GPRC5D-AS1 in the cells in various groups were detected by RT-qPCR method.The levels of reactive oxygen species(ROS),glutathione peroxidase 4(GPX4),ferrous ion(Fe2+)and malondialdehyde(MDA)in the cells in various groups were detected by flow cytometry and enzyme-linked immunosorbent assay(ELISA)method;the ultrastructural changes of mitochondria in the cells in various groups were observed under transmission electron microscope;the expression of myogenic differentiation protein(MyoD)in the cells in various groups was detected by immunofluorescence;the expression levels of ferroptosis-related proteins in the cells in various groups were detected by Western blotting method.Results:The best modeling effect was achieved with 100 mg·L^(-1) dexamethasone at 48 h.Compared with normal C2C12 cells,the expression level of lncRNA GPRC5D-AS1 in the C2C12 cells after treated with 100 mg·L^(-1) dexamethasone for 48 h was decreased(P<0.05),confirming that lncRNA GPRC5D-AS1 had a regulatory role in the muscle atrophy model.The RT-qPCR results showed that compared with normal group,the expression level of lncRNA GRPC5D-AS1 in the cells in model group was decreased(P<0.05);compared with model group and lncRNA GRPC5D-AS1-NC group,the expression le

关 键 词：肌萎缩 长链非编码RNA GPRC5D-AS1 铁死亡 地塞米松 成肌细胞 

分 类 号：R685[医药卫生—骨科学]