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

作　　者：林莉 李菊霜 朱戈丽 张艳霞 伍军 LIN Li;LI Ju-shuang;ZHU Ge-li;ZHANG Yan-xia;WU Jun(Department of Nephrology,Wuhan Third Hospital(Tongren Hospital of Wuhan University),Wuhan 430074,China)

机构地区：[1]武汉市第三医院肾内科,武汉430074

出　　处：《微循环学杂志》2024年第3期1-5,11,共6页Chinese Journal of Microcirculation

基　　金：湖北省自然科学基金(2016CFB590);武汉市卫健委科研项目(WX16B09)。

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、IL-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。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 aspartate 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 respectively 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-1βexpression 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-1βexpression and activity of Caspase-1 were significantly lower in miR-22-3p mimic+LPS+ATP group but significantly higher in miR-22-3p inhibitor+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 NLRP3 

分 类 号：R692.6[医药卫生—泌尿科学]