miR-223下调NLRP3炎性体表达抑制人血管平滑肌细胞分泌高迁移率族蛋白B1

MiR-223 inhibits HMGB1 secretion from human arterial smooth muscle cells by down-regulating NLRP3

目的探讨microRNA-223(miR-223)下调人动脉平滑肌细胞(human arterial smooth muscle cells,HASMCs)高迁移率族蛋白B1(high mobility group box 1,HMGB1)分泌的作用机制。方法利用我院严重外伤截肢患者或健康器官捐献者的正常动脉组织,通过组织块贴壁法分离原代HASMCs并通过免疫荧光进行鉴定;利用实时荧光定量PCR(real-time quantitative PCR,RT-q PCR)及Western blot检测脂多糖(lipopolysaccharides,LPS)联合腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)对HASMCs中NLRP3(NLR family pyrin domain containing 3)炎性体的激活作用;转染miR-223模拟物(miR-223mimic)进入HASMCs,利用RT-q PCR、Western blot检测靶基因NLRP3 mRNA、蛋白表达量及激活状态,利用双荧光素酶报告基因明确miR-223和NLRP3的调控关系,通过酶联免疫吸附测定法(ELISA)检测miR-223对HASMCs上清中HMGB1的含量影响。结果 LPS联合ATP可以激活HASMCs细胞内NLRP3炎性体(P<0.05),促进HMGB1胞外分泌(P<0.01);转染miR-223可以显著减少NLRP3mRNA及蛋白水平(P<0.05),通过双荧光素酶报告基因明确NLRP3为miR-223的直接靶基因;转染miR-223可以明显下调HASMCs上清的HMGB1水平(P<0.01)。结论 miR-223可以通过转录后水平下调HASMCs内NLRP3炎性体的表达及激活,从而抑制HASMCs分泌HMGB1。

Objective To investigate the role and mechanism of microRNA-223 （miR-223） in down-regulation of high mobility group box 1 （ HMGB1 ） secretion from human arterial smooth muscle cells （HASMCs）. Methods HASMCs were isolated from the normal arteries of severe traumatic amputees or healthy donors without arteriosclerosis obliterans by tissue explants adherent and then identified by immunofluorescence assay of SM-α actin. Real-time quantitative PCR （RT-qPCR） and Western blotting were used to detect the activity of NLR family pyrin domain containing 3 （ NLRP3 ） inflammasome in the HASMCs after treatment of lipopolysaccharides （LPS） and adenosine triphosphate （ATP）. After transfection of miR- 223 mimic, the expression of NLRP3 was detected through RT-qPCR and Western blotting. Dual-luciferase report system was performed to verify the regulatory relationships between miR-223 and NLRP3 after the 3＇UTR mutation site of NLRP3 was designed. The level of HMGB1 in the supernatant of transfected HASMCs was measured by enzyme linked immunosorbent assay （ELISA）. Results Treatment of LPS and ATP enhanced the activity of NLRP3 inflammasome （ P 〈 0.05 ） , and improved the secretion of HMGB1 from the HASMCs （P 〈 0.05 ）. Transfection of miR-223 mimic significantly down-regulated NLRP3 at both mRNA and protein levels （P 〈0.05）. Dual-luciferase report system indicated that NLRP3 was the direct target of miR- 223 because of its mRNA 3＇-UTR sequences being complementary to miR-223. HMGBI secretion from HASMCs was remarkably decreased after transfection with miR-223 mimic （P 〈 0.01）. Conclusion miR- 223 can decreases expression of NLRP3 inflammasome at transcriptional level and then inhibit its activity, and subsequently suppress the secretion of HMGB1 from HASMCs.