微小RNA-22-3p抑制人肾小管上皮细胞NLRP3活化
MicroRNA-22-3p Inhibits NLRP3 Activation in Human Renal Tubular Epithelial Cells
林莉 1李菊霜 1朱戈丽 1张艳霞 1伍军1
作者信息
- 1. 武汉市第三医院肾内科,武汉 430074
- 折叠
摘要
目的:探讨微小RNA-22-3p(miR-22-3p)对人肾小管上皮细胞NLRP3活化的影响.方法:将包含NLRP3基因3'-非编码区(3'-UTR)序列的野生型及突变型双荧光素酶报告基因质粒(psiCHECK2)分别与miR-22-3p mimic(模拟物)、miR-22-3p inhibitor(抑制物)共转染人肾小管上皮细胞株(HK-2),检测细胞荧光素酶活性;HK-2 细胞随机分组如下:(1)正常对照组;(2)miR-22-3p mimic+LPS+ATP 组;(3)miR-22-3p control+LPS+ATP 组;(4)miR-22-3p inhibitor+LPS+ATP 组;(5)miR-22-3p mimic+LPS 组;(6)miR-22-3p control+LPS 组;(7)miR-22-3p inhibitor+LPS组,采用定量PCR(RT-PCR)、免疫印迹(WB)分别检测NLRP3 mRNA和蛋白的表达,酶联免疫吸附法(ELISA)分别检测白介素-1β(IL-1β)水平及半胱天冬氨酸蛋白酶-1(Caspase-1)活性.结果:NLRP3-3'UTR野生型分别与miR-22-3p mimic、miR-22-3p inhibitor共转染HK-2细胞后,与对照组比较,荧光素酶活性分别出现降低与升高(P＜0.05).此外,与miR-22-3p control+LPS组比较,miR-22-3p mimic+LPS组NLRP3 mRNA 和蛋白表达、1L-1β 水平及 Caspase-1 活性均下降(P＜0.05),miR-22-3p inhibitor+LPS 组 NLRP3 mRNA 和蛋白表达、IL-1β 水平及 Caspase-1 活性均升高(P＜0.05);与 miR-22-3p control+LPS+ATP 组比较,miR-22-3p mimic+LPS+ATP 组 NLRP3 mRNA 和蛋白表达、IL-1β 水平及 Caspase-1 活性均下降(P＜0.05),miR-22-3p inhibitor+LPS+ATP 组 NLRP3 mRNA 和蛋白表达、IL-1β 水平及 Caspase-1 活性均升高(P＜0.05).结论:miR-22-3p可抑制人肾小管上皮细胞NLRP3活化.
Abstract
Objective:To explore the role of microRNA-22-3p(miR-22-3p)in modulating NLRP3(NACHT,LRR and PYD domains-containing protein 3)in human renal tubular epithelial cells.Method:The dual-luciferase reporter plasmid(psiCHECK2)containing the wild and mutant type of NLRP3-3'UTR(untranslated regions),miR-22-3p mimic and miR-22-3p inhibitor were transfected into the human renal tubular epithelial cell line(termed HK-2)respectively.And the luciferase activity was evaluated in HK-2 cells.HK-2 cells were grouped randomly:(1)normal control;(2)miR-22-3p mimic plus lipopolysaccharide(LPS)plus adenosine triphosphate(ATP);(3)miR-22-3p control plus LPS plus ATP;(4)miR-22-3p inhibitor plus LPS plus ATP;(5)miR-22-3p mimic plus LPS;(6)miR-22-3p control plus LPS;(7)miR-22-3p inhibitor plus LPS.The expression of NLRP3 was evaluated by real-time polymerase chain reaction(RT-PCR)and western blot(WB).Enzyme-linked immunosorbent assay(ELISA)was used for evaluation of IL-1β(interleukin-1beta)expression and for assaying the activity of cysteine-requiring as-partate protease-1(Caspase-1).Results:NLRP3 wild-type recombinant plasmid and miR-22-3p mimic or miR-22-3p inhibitor were transfected into HK-2 cells,the luciferase activity was significantly decreased or increased respective-ly when compared to the control group(P＜0.05).Compared with miR-22-3p control+LPS group,the mRNA and protein expressions of NLRP3,IL-1β expression and activity of Caspase-1 were significantly decreased in miR-22-3p mimic+LPS group but significantly increased in miR-22-3p inhibitor+LPS group(P＜0.05);Compared with miR-22-3p control+LPS+ATP group,the mRNA and protein expressions of NLRP3,IL-1β expression and activity of Caspase-1 were significantly lower in miR-22-3p mimic+LPS+ATP group but significantly higher in miR-22-3p in-hibitor+LPS+ATP group(P＜0.05).Conclusion:MicroRNA-22-3p can inhibit NLRP3 activation in human renal tubular epithelial cells.
关键词
miR-22-3p/人肾小管上皮细胞/NLRP3Key words
miR-22-3p/Human renal tubular epithelial cells/NLRP3引用本文复制引用
基金项目
湖北省自然科学基金(2016CFB590)
武汉市卫健委科研项目(WX16B09)
出版年
2024
NETL
NSTL
万方数据