Xue Xin, Qiu Yi, Yang Hong-Li
Department of Pathogen Biology & Immunology, Xi'an Jiaotong University Health Science center, Xi'an, China.
The Fourth internal medicine, Xi'an Chest Hospital, Xi'an, China.
Cell Physiol Biochem. 2017;42(1):91-102. doi: 10.1159/000477118. Epub 2017 May 11.
BACKGROUND/AIMS: The purpose of this study is to explore the immunoregulatory role of microRNA-21 (miR-21) targeting of the TLR4/MyD88 signaling pathway in macrophages in response to Bacillus Calmette-Guerin (BCG) infection.
After infection with BCG, mouse RAW246.7 cells were assigned into control, BCG, miR-21 mimic + BCG, mimic-negative control (NC) + BCG, miR-21 inhibitor + BCG, inhibitor-NC + BCG, BCG + TAK242 (an inhibitor of the TLR4 signaling pathway), and miR-21 inhibitor + TAK242 + BCG groups. Western blotting and qRT-PCR were used to detect the expression of miR-21, TLR4 and MyD88. The levels of TNF-a, IL-6 and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA). Cell viability was measured using an MTT assay. Cell apoptosis and necrosis rates were detected using flow cytometry.
Compared with the control group, miR-21 expression and levels of TNF-a, IL-6 and IL-10, as well as cell apoptosis and necrosis rates, were elevated, while expression of TLR4 and MyD88, as well as cell viability, were reduced in BCG infection groups. Compared with the BCG group, miR-21 expression was increased in the miR-21 mimic + BCG group but decreased in the miR-21 inhibitor + BCG and miR-21 inhibitor + TAK242 + BCG groups. The expression of TLR4 and MyD88, as well as the cell viability, were decreased, while levels of TNF-a, IL-6 and IL-10, as well as cell apoptosis and necrosis rates, were increased in the miR-21 mimic + BCG and TAK242 + BCG groups. The opposite trends were found in the miR-21 inhibitor + BCG group. Compared with the TAK242 + BCG group, the miR-21 inhibitor + TAK242 + BCG group had higher expression of TLR4 and MyD88 as well as higher cell viability and lower levels of TNF-a, IL-6, IL-10, cell apoptosis and necrosis rates. However, the miR-21 inhibitor + TAK242 + BCG group exhibited the opposite trends when compared with the miR-21 inhibitor + BCG group.
Our results suggest that miR-21 can negatively modulate the TLR4/MyD88 signaling pathway, resulting in decreased cell viability, increased cell apoptosis and increased levels of inflammatory factors following BCG infection in macrophages.
背景/目的：本研究旨在探讨微小RNA-21（miR-21）靶向Toll样受体4（TLR4）/髓样分化因子88（MyD88）信号通路在巨噬细胞应对卡介苗（BCG）感染时的免疫调节作用。
用BCG感染后，将小鼠RAW246.7细胞分为对照组、BCG组、miR-21模拟物+BCG组、模拟物阴性对照（NC）+BCG组、miR-21抑制剂+BCG组、抑制剂-NC+BCG组、BCG+TAK242（TLR4信号通路抑制剂）组以及miR-21抑制剂+TAK242+BCG组。采用蛋白质免疫印迹法和定量逆转录聚合酶链反应（qRT-PCR）检测miR-21、TLR4和MyD88的表达。采用酶联免疫吸附测定（ELISA）检测肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-10（IL-10）的水平。采用MTT法检测细胞活力。采用流式细胞术检测细胞凋亡率和坏死率。
与对照组相比，BCG感染组中miR-２1表达、TNF-α、IL-6和IL-10水平以及细胞凋亡率和坏死率升高，而TLR4和MyD88的表达以及细胞活力降低。与BCG组相比，miR-21模拟物+BCG组中miR-21表达增加，而miR-21抑制剂+BCG组和miR-21抑制剂+TAK242+BCG组中miR-21表达降低。miR-21模拟物+BCG组和TAK242+BCG组中TLR4和MyD88的表达以及细胞活力降低，而TNF-α、IL-6和IL-10水平以及细胞凋亡率和坏死率升高。miR-21抑制剂+BCG组则呈现相反趋势。与TAK242+BCG组相比，miR-21抑制剂+TAK242+BCG组中TLR4和MyD88的表达更高，细胞活力更高，TNF-α、IL-6、IL-10水平、细胞凋亡率和坏死率更低。然而，与miR-21抑制剂+BCG组相比，miR-21抑制剂+TAK242+BCG组呈现相反趋势。
我们的结果表明，miR-21可负向调节TLR4/MyD88信号通路，导致巨噬细胞在BCG感染后细胞活力降低、细胞凋亡增加以及炎症因子水平升高。
