miR-485-5p通过靶向白介素-1受体关联激酶4调控巨噬细胞炎症反应  

miR-485-5p regulates the inflammatory response of macrophages by targeting interleukin-1 receptor-associated kinase 4

作　　者：郑锡铭 徐雪勤 张爱荣 杨喜永 赵小燕 陈仪 ZHENG Xi-ming;XU Xue-qin;ZHANG Ai-rong;YANG Xi-yong;ZHAO Xiao-yan;CHEN Yi(Department of Medical Laboratory,Zhumadian Central Hospital,Henan 463000,China)

机构地区：[1]驻马店市中心医院医学检验科,河南463000

出　　处：《中国卫生检验杂志》2023年第22期2761-2765,共5页Chinese Journal of Health Laboratory Technology

摘　　要：目的探究miR-485-5p对白介素-1受体关联激酶4(IRAK-4)的靶向作用,研究其在巨噬细胞模型炎症反应中的调控作用。方法合成miR-485-5p模拟物(mimics)及其抑制剂(inhibitors),分别转染HEK-293T细胞,RT-PCR检测miR-485-5p的表达水平。根据miRNA靶向作用预测数据分析选定IRAK-4作为研究的靶基因,构建IRAK-43'UTR及其突变体萤火虫-海肾双荧光素酶报告基因系统,通过检测在细胞模型中过表达/抑制表达miR-485-5p后对荧光素酶活力的影响,验证miR-485-5p与IRAK-4之间的靶向关系,Westernblot检测IRAK-4蛋白质表达水平。构建巨噬细胞模型,将miR-485-5pmimics及inhibitors分别转染巨噬细胞,RT-PCR检测靶基因IRAK-4mRNA的表达水平,ELISA检测上清中下游致病因子IL-6、IL-17、IL-18的表达。结果通过实验证实miR-485-5p与IRAK-4存在靶向作用。细胞模型中,与对照组相比,转染miR-485-5pmimics后miR-485-5p表达水平升高了17.15倍(P<0.01),转染miR-485-5pinhibitors后miR-485-5p表达水平降低了79.80%(P<0.05)。过表达miR-485-5p后明显抑制IRAK-4的蛋白水平表达及mRNA相对含量,抑制下游炎性因子IL-6、IL-17、IL-18的表达(P<0.05);抑制表达miR-485-5p,则上调IRAK-4的蛋白水平及mRNA相对含量,可显著提高IL-6、IL-17、IL-18的释放(P<0.05)。结论证实miR-485-5p可以通过对靶向IRAK-4的特异性序列,调控下游炎性细胞因子的表达,进而调控细胞的炎症反应。Objective This paper aims to investigate the targeting effect of miR-485-5p on interleukin-1 receptor associat-ed kinase 4(IRAK-4)and its regulatory role in the inflammatory response of macrophage model.Methods miR-485-5p mimics and their inhibitors were synthesized and transfected into HEK-293T cells.The expression of miR-485-5p was detected by RT-PCR.IRAK-4 was selected as the target gene for the study according to the analysis of miRNA targeting effect prediction data,and IRAK-43UTR and its mutant firefly-Renilla dual luciferase reporter gene system were construc-ted.The targeting relationship between miR-485-5p and IRAK-4 was verified by detecting the effect of overexpression/inhi-bition of miR-485-5p on luciferase activity in the cell model,and the protein expression of IRAK-4 was detected by West-ern blot.The macrophage model was constructed,and miR-485-5p mimics and inhibitors were transfected into macrophages,respectively.The expression of target gene IRAK-4 mRNA was detected by RT-PCR,and the expression of pathogenic factors IL-6,IL-17 and IL-18 in the supernatant was detected by enzyme-linked immunosorbent assay(ELISA).Results It was confirmed by experiments that miR-485-5p and IRAK-4 had a targeted effect.In the cell model,compared with the control group,the expression of miR-485-5p after transfection of miR-485-5p mimics increased by 17.15 times(P<0.01),and the expression of miR-485-5p after transfection of miR-485-5p inhibitors decreased by 79.80%(P<0.05).Over-expression of miR-485-5p significantly inhibited the protein level and mRNA relative content of IRAK-4 and the expression of downstream inflammatory factors,IL-6,IL-17 and IL-18(P<0.05).Inhibiting the expression of miR-485-5p up-regulated the protein level and relative mRNA content of IRAK-4 and significantly increased the release of IL-6,IL-17,and IL-18(P<0.05).Conclusion miR-485-5p can regulate the expression of downstream inflammatory cytokines by targeting the specific sequence of IRAK-4,and then regulate the inflammatory response of cells.

关 键 词：miR-485-5p 白介素-1受体关联激酶4 人单核细胞白血病细胞系 

分 类 号：R446[医药卫生—诊断学]