miR-221对结核分枝杆菌感染巨噬细胞后炎性因子表达的影响

Effects of miR-221 on the inflammatory factor secretion from macrophages after Mycobacterium tuberculosis infection

目的探讨microRNA-221(miR-221)对结核分枝杆菌感染巨噬细胞后炎症因子水平的影响。方法用qRT-PCR检测结核分枝杆菌感染后巨噬细胞中miR-221的表达;用miR-221模拟物和miR-221抑制物转染结核分枝杆菌感染的巨噬细胞,采用酶联免疫吸附测定(ELISA)法和Griess法分别检测炎症因子表达和NO分泌;双荧光素酶报告基因、q RT-PCR和Western blot法检测miR-221和Rho相关蛋白激酶1(ROCK1)的靶向关系。结果 miR-221在结核分枝杆菌感染的巨噬细胞中低表达;miR-221模拟物上调巨噬细胞miR-221的表达水平,抑制巨噬细胞肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β、IL-6和NO的分泌;miR-221抑制物下调巨噬细胞miR-221的表达水平,促进巨噬细胞TNF-α、IL-1β、IL-6和NO的分泌;miR-221可作用于ROCK1的3′UTR,并抑制其表达(P<0.05)。结论 miR-221通过靶向抑制ROCK1的表达抑制结核分枝杆菌感染巨噬细胞后的炎症因子的表达。

Objective To explore the effects of miR-221 on the inflammatory factor secretion from macrophages after Mycobacterium tuberculosis infection. Methods The expression of miR-221 in macrophages after Mycobacterium tuberculosis infection was detected by real-time quantitative PCR（q RT-PCR）. Macrophages were transiently transfected with miR-221 mimic or miR-221 inhibitor. The expression of macrophage inflammatory factor and secretion of NO were detected by enzyme-linked immunosorbent assay（ELISA） and Griess method, respectively. The relationship of miR-221 and Rho-associated, coiled-coil containing protein kinase 1（ROCK1） was analyzed by dual-luciferase reporter gene assay, q RT-PCR and Western blot. Results Down-regulated miR-221 was observed in macrophages infected with Mycobacterium tuberculosis. The expression of miR-221 in macrophages was up-regulated by miR-221 mimics, but down-regulated by miR-221 inhibitors. The secretions of TNF-α, IL-1β, IL-6 and NO were significantly accelerated in macrophages infected with Mycobacterium tuberculosis, and increased in miR-221 up-regulated macrophages, but suppressed in miR-221 down-regulated macrophages. miR-221 directly binds to the 3＇ UTR of ROCK1, and negatively regulated its expression. Conclusion miR-221 can regulate the ROCK1 expression and inhibit the secration of inflammatory factors expression in macrophages after Mycobacterium tuberculosis infection.