miR-663b对IL-1β诱导的髓核细胞炎症反应和凋亡的影响及其机制  

EFFECT OF miR-663b ON INTERLEUKIN-1β-INDUCED INFLAMMATORY RESPONSE AND APOPTOSIS OF NUCLEUS PULPOSUS CELLS AND ITS MECHANISM

作　　者：李亚雄 马学晓[1] 常胜 魏嘉豪 刘勇[1] LI Yaxiong;MA Xuexiao;CHANG Sheng;WEI Jiahao;LIU Yong(Spine Surgery Department,The Affiliated Hospital of Qingdao University,Qingdao 266035,China)

机构地区：[1]青岛大学附属医院脊柱外科,山东青岛266035

出　　处：《精准医学杂志》2023年第3期210-214,218,共6页Journal of Precision Medicine

摘　　要：目的 探讨miR-663b对白细胞介素-1β(IL-1β)诱导的髓核细胞(NPCs)炎症反应和凋亡的影响及其机制。方法 根据对NPCs不同处理方式分为A组(无任何处理)、B组(IL-1β诱导)、C组(IL-1β诱导+miR-663b mimic转染)、D组(IL-1β诱导+miR-633b NC转染)。采用实时荧光定量PCR(RT-qPCR)法检测4组NPCs中miR-663b及炎症因子(TNF-α、IL-6、IL-1β)、Ⅱ型胶原蛋白、多聚糖蛋白基因的表达情况,采用CCK8法和TUNEL染色法检测4组NPCs增殖和凋亡情况,采用免疫印迹法检测各组NPCs IL-1受体1(IL1R1)蛋白表达情况。使用TargetScan数据库预测miR-663b与IL1R1间的潜在结合位点,将293T细胞分E组(转染IL1R1-wt质粒+miR-663b mimic)、F组(转染IL1R1-wt质粒+miR-663b mimic NC)、G组(转染IL1R1-mut质粒+miR-663b mimic)和H组(转染IL1R1-mut质粒+miR-663b mimic NC),检测各组NPCs荧光素酶的活性。结果 RT-qPCR结果显示,C组较A、B、D组miR-663b相对表达量显著上升(t=9.41~22.93,P<0.01);与B、D组相比,C组TNF-α、IL-6、IL-1β、Ⅱ型胶原蛋白及多聚糖蛋白基因相对表达量显著改变(t=3.17~32.51,P<0.01)。CCK8法及TUNEL染色法结果显示,与B、D组相比,C组NPCs增殖显著增加(t=3.14、3.96,P<0.01),而细胞凋亡显著减少(t=4.28、168.61,P<0.01)。RT-qPCR和免疫印迹法结果显示,与B、D组相比,C组NPCs IL1R1和IL1R1蛋白相对表达量显著下降(t=6.39~12.84,P<0.01)。双荧光素酶报告基因实验显示,E组荧光素酶活性显著低于F、G、H组(t=10.62~16.27,P<0.01)。结论 miR-663b或通过与IL1R1靶向结合下调NPCs的IL1R1表达,并减缓NPCs炎症反应及凋亡进程。Objective To investigate the effect of miR-663b on the inflammatory response and apoptosis of nucleus pulposus cells(NPCs)induced by interleukin-1β(IL-1β)and its mechanism.Methods According to the different treatment me-thods,NPCs were divided into group A(no treatment),group B(induced by IL-1β),group C(IL-1βinduction+miR-663b mimic transfection),and group D(IL-1βinduction+miR-633b NC transfection).RT-qPCR was used to measure the expression levels of miR-663b,inflammatory factors\,typeⅡcollagen,and po-lysaccharide in NPCs;CCK-8 assay and TUNEL staining were used to observe the proliferation and apoptosis of NPCs;Western blotting was used to measure the protein expression level of interleukin-1 receptor 1(IL1R1)in NPCs.TargetScan database was used to predict the potential binding sites between miR-663b and IL1R1.The 293T cells were divided into group E(transfected with IL1R1-wt plasmid+miR-663b mimic),group F(transfected with IL1R1-wt plasmid+miR-663b mimic),group G(transfected with IL1R1-mut plasmid+miR-663b mimic),and group H(transfected with IL1R1-mut plasmid+mimic NC),and the luciferase activity of NPCs was measured for each group.Results RT-qPCR results showed that compared with groups A,B,and D,group C had a significant increase in the relative expression level of miR-663b(t=9.41-22.93,P<0.01),and compared with groups B and D,group C had significant changes in the relative expression levels of TNF-α,IL-6,IL-1β,typeⅡcollagen,and polysaccharide(t=3.17-32.51,P<0.01).CCK-8 assay and TUNEL staining showed that compared with groups B and D,group C had a significant increase in the proliferation of NPCs(t=3.14,3.96,P<0.01)and a significant reduction in the apoptosis of NPCs(t=4.28,168.61,P<0.01).RT-qPCR and Western blotting showed that compared with groups B and D,group C had signi-ficant reductions in the relative protein expression levels of IL1R1 and IL1R1 in NPCs(t=6.39-12.84,P<0.01).Dual-luciferase reporter assay showed that group E had a significantly lower luciferase activity than groups F,G,and H

关 键 词：微RNAS 髓核 基因表达调控 细胞增殖 细胞凋亡 受体 白细胞介素1 椎间盘退行性变 

分 类 号：R681.5[医药卫生—骨科学]