miR-613调控SphK1/S1P通路对IL-1β诱导的软骨细胞损伤的影响OA
The Effect of miR-613 on IL-1β-Induced Chondrocyte Injury by Regulating the SphK1/S1P Pathway
目的:探讨微小 RNA-613(miR-613)调控鞘氨醇激酶1(SphK1)/鞘氨醇-1-磷酸(S1P)通路对白细胞介素-1β(IL-1β)诱导的软骨细胞损伤的影响.方法:双荧光素酶报告基因实验检测 miR-613 与 SphK1 的互作;将 CHON-001 细胞分为 Ctrl 组(正常培养)、IL-1β 组(10ng/mL 的 IL-1β 诱导)、NC-mimics 组(转染 NC-mimics+IL-1β 诱导)、miR-613-mimics 组(转染 miR-613-mimics+IL-1β诱导)、miR-613-mimics+OE-NC 组(转染 miR-613-mimics+OE-NC+IL-1β 诱导)、miR-613-mimics+OE-SphK1 组(转染 miR-613-mimics+OE-SphK1+IL-1β 诱导).qRT-PCR 法检测 CHON-001 细胞中miR-613、SphK1、S1P mRNA 表达;MTT 和 EdU 染色检测 CHON-001 细胞增殖;流式细胞仪和 TUNEL染色检测 CHON-001 细胞凋亡;ELISA 试剂盒检测 CHON-001 细胞中 IL-6、TNF-α 的表达;Western blot 检测 CHON-001 细胞中 SphK1、S1P、MMP-13、ADAMTS-5 蛋白表达.结果:miR-613 可以靶向负调控 SphK1.与 Ctrl 组相比,IL-1β 组 EdU 阳性细胞率、OD570 值、miR-613 降低,凋亡率、IL-6、TNF-α、SphK1 mRNA 和蛋白、S1P mRNA 和蛋白、MMP-13、ADAMTS-5 升高(P<0.05);与 IL-1β 组、NC-mimics 组相比,miR-613-mimics 组 EdU 阳性细胞率、OD570 值、miR-613 升高,凋亡率、IL-6、TNF-α、SphK1 mRNA 和蛋白、S1P mRNA 和蛋白、MMP-13、ADAMTS-5 降低(P<0.05);与 miR-613-mimics组、miR-613-mimics+OE-NC 组相比,miR-613-mimics+OE-SphK1 组 EdU 阳性细胞率、OD570 值降低,凋亡率、IL-6、TNF-α、SphK1 mRNA 和蛋白、S1P mRNA 和蛋白、MMP-13、ADAMTS-5 升高(P<0.05).结论:miR-613 可能通过抑制 SphK1/S1P 通路抑制 IL-1β 诱导的软骨细胞损伤.
Objective:To explore the effect of microRNA-613(miR-613)on interleukin-1β(IL-1β)-induced chondrocyte injury by regulating the sphingosine kinase 1(SphK1)/sphingosine 1-phosphate(S1P)pathway.Methods:Dual luciferase reporter gene assay was used to detect the interaction between miR-613 and SphK1.CHON-001 cells were divided into six groups:Ctrl group(normal culture),IL-1β group(10ng/mL IL-1β induction),NC-mimics group(NC-mimics transfection+IL-1β induction),miR-613-mimics group(miR-613-mimics transfection+IL-1β induction),miR-613-mimics+OE-NC group(miR-613-mimics+OE-NC co-transfection+IL-1β induction),and miR-613-mimics+OE-SphK1 group(miR-613-mimics+OE-SphK1 co-transfection+IL-1β induction).QRT-PCR was used to detect the expression of miR-613,SphK1,and S1P mRNA in CHON-001 cells.MTT and EdU staining were used to measure CHON-001 cell proliferation.Flow cytometry and TUNEL staining were performed to measure CHON-001 cell apopto-sis.An ELISA kit was used to measure the expression of IL-6 and TNF-α in CHON-001 cells.Moreover,a Western blot was performed to detect the expression of SphK1,S1P,MMP-13,and ADAMTS-5 proteins in CHON-001 cells.Results:MiR-613 could target the negative regulation of SphK1.Compared to the Ctrl group,the IL-1β group showed decreased EdU-positive cell rate,OD570 value,and miR-613 expression,while apoptosis rate,levels of IL-6,TNF-α,SphK1 mRNA and protein,S1P mRNA and protein,MMP-13,and ADAMTS-5 were increased(P<0.05).Compared to the IL-1β group and the NC-mimics group,the miR-613-mimics group exhibited increased EdU-positive cell rate,OD570 value,and miR-613 expression,whereas the apoptosis rate,levels of IL-6,TNF-α,SphK1 mRNA and protein,S1P mRNA and protein,MMP-13,and ADAMTS-5 were decreased(P<0.05).Compared to the miR-613-mimics group and the miR-613-mimics+OE-NC group,the miR-613-mimics+OE-SphK1 group had decreased EdU-positive cell rate and OD570 value,while apoptosis rate,levels of IL-6,TNF-α,SphK1 mRNA and protein,S1P mRNA and pro-tein,MMP-13,and ADAMTS-5 were increased(P<0.05).Conclusion:MiR-613 may inhibit IL-1β-in-duced chondrocyte injury by suppressing the SphK1/S1P pathway.
张佚舟;孙承军
武汉科技大学附属华润武钢总医院骨科,湖北 武汉 430080||武汉科技大学医学院医学部,湖北 武汉 430080武汉科技大学附属华润武钢总医院骨科,湖北 武汉 430080
软骨细胞损伤微小RNA-613鞘氨醇激酶1鞘氨醇-1-磷酸白细胞介素-1β
Chondrocyte injuryMicroRNA-613Sphingosine kinase 1Sphingosine-1-phos-phateInterleukin-1β
《河北医学》 2026 (4)
542-549,8
湖北省武汉市医学科研项目,(编号:WX21D83)
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