miR-22靶向NLRP3在体外小鼠巨噬细胞RAW264.7细胞系中清除结核分枝杆菌

miR-22 clears Mycobacterium tuberculosis from mouse macrophage RAW264.7 cell line in vitro by targeting NLRP3

目的研究微小RNA-22(miR-22)靶向核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)在巨噬细胞RAW264.7清除结核分枝杆菌(MTB)中的作用。方法体外培养RAW264.7巨噬细胞,分为miR-22 mimic+NLRP3-WT组、miR-22 NC+NLRP3-WT组、miR-22 mimic+NLRP3-MUT组和miR-22 NC+NLRP3-MUT组。采用双荧光素酶报告基因实验验证miR-22与NLRP3基因的靶向关系。采用MTB标准菌株H37Rv感染RAW264.7巨噬细胞,分为NC组(正常生长RAW264.7巨噬细胞)、H37Rv组(感染H37Rv的RAW264.7巨噬细胞)、H37Rv+mimic NC组(H37Rv组基础上转染miR-22 mimic NC序列)和H37Rv+mimic组(H37Rv组基础上转染miR-22 mimic序列),采用流式细胞仪检测RAW264.7巨噬细胞微管相关蛋白轻链3-II(LC3-II)+、MTB抗原Ag85A+(Ag85A+);采用实时定量PCR(RT-qPCR)检测RAW264.7巨噬细胞中miR-22、NLRP3 mRNA表达水平;采用免疫印迹(WB)检测RAW264.7巨噬细胞中NLRP3、半胱天冬氨酸蛋白酶-1(caspase-1)蛋白表达水平;酶联免疫吸附试验(ELISA)检测RAW264.7巨噬细胞上清液中白细胞介素-1β(IL-1β)、IL-18水平。结果双荧光素酶报告基因实验结果显示,与miR-22 NC+NLRP3-WT组比较,miR-22 mimic+NLRP3-WT组荧光素酶相对活性显著降低(P<0.05);miR-22 mimic+NLRP3-MUT组、miR-22 NC+NLRP3-MUT组荧光素酶相对活性差异无统计学意义(P>0.05)。与NC组比较,H37Rv组RAW264.7巨噬细胞miR-22水平、LC3-II+细胞比例显著降低(P<0.05),Ag85A+细胞比例、NLRP3水平、caspase-1、IL-1β、IL-18水平显著增加(P<0.05);与H37Rv+mimic NC组比较,H37Rv+mimic组RAW264.7巨噬细胞miR-22水平、LC3-II+细胞比例增加(P<0.05),Ag85A+细胞比例、NLRP3水平、caspase-1、IL-1β、IL-18水平显著降低(P<0.05)。结论 miR-22通过靶向抑制NLRP3表达,促进巨噬细胞清除结核分枝杆菌,可能与促进细胞自噬有关。

This study was performed to investigate the role of microRNA-22(miR-22) targeting nucleotidebinding oligomerization domain-like receptor protein 3(NLRP3) in the clearance of Mycobacterium tuberculosis(MTB) from macrophage RAW264.7. RAW264.7 macrophages were cultured in vitro and divided into miR-22 mimic+NLRP3-WT group, miR-22 NC+NLRP3-WT group, miR-22 mimic+NLRP3-MUT group and miR-22 NC+NLRP3-MUT group. The targeting relationship between miR-22 and NLRP3 gene was verified by double luciferase reporter gene experiment. The standard MTB strain H37 Rv was used to infect RAW264.7 macrophages, which were divided into NC group(normal grew RAW264.7 macrophages), H37 Rv group(RAW264.7 macrophages infected with H37 Rv), H37 Rv+mimic NC group(transfected with miR-22 mimic NC sequence on the basis of H37 Rv group) and H37 Rv + mimic group(transfected with miR-22 mimic sequence on the basis of H37 Rv group). Flow cytometry was used to detect the microtubule associated protein light chain 3-II(LC3-II)+and MTB antigen Ag85A^+(Ag85A^+) of RAW264.7 macrophages;the expression levels of miR-22 and NLRP3 mRNA in RAW264.7 macrophages were detected by RT-qPCR;the protein expression levels of NLRP3 and caspase-1 in RAW264.7 macrophages were detected by Western blotting;and the levels of IL-1βand IL-18 in the supernatant of RAW264.7 macrophage were detected by ELISA. The results of double luciferase reporter gene experiment showed that the relative activity of luciferase in miR-22 mimic + NLRP3-WT group decreased significantly compared with miR-22 NC + NLRP3-WT group(P<0.05), while there was no significant difference in luciferase activity between miR-22 mimic+NLRP3-MUT group and miR-22 NC+NLRP3-MUT group(P>0.05). Compared with NC group, H37 Rv group demonstrated lower level of miR-22 and lower proportion of LC3-II^+cells(P<0.05), but higher proportion of Ag85A^+cells, higher levels of NLRP3, caspase-1, IL-1β and IL-18(P<0.05);compared with H37 Rv + mimic NC group, the level of miR-22 and the proportion of LC3-II^+cells in RAW264.7 macrophages of H37 Rv+mimic NC group increased(P<0.05), and the proportion of Ag85 A+cells, the levels of NLRP3, caspase-1, IL-1β, IL-18 decreased significantly(P<0.05). In conclusion, miR-22 can promote macrophages to eliminate Mycobacterium tuberculosis by targeting NLRP3 expression, and the mechanism may relate to autophagy promotion.