miRNA-146a在小鼠小胶质细胞炎症反应中的作用

Role of microRNA-146a in inflammatory responses in microglial cells of mice

目的评价microRNA(miR)-146a在小鼠小胶质细胞炎症反应中的作用。方法小鼠小胶质细胞BV2进行培养,选取对数生长期的细胞,采用随机数字表法分为5组(n=48):对照组(C组)、LPS组、LPS+miR-146a模拟物组(LPS-M组)、LPS+miR146-a抑制物组(LPS-I组)和LPS+阴性对照组(LPS-NC组)。LPS-M组、LPS-I组和LPS-NC组分别将miR-146-a模拟物RNA、miR-146a抑制物RNA和模拟物/抑制物的错义RNA转染至细胞。转染成功后,LPS组、LPS-M组、LPS-I组和LPS-NC组细胞分别加入终浓度为1μg/ml的LPS,C组加入等容量培养基,孵育6 h。采用qRT-PCR法检测细胞miR-146a、白介素受体相关激酶1(IRAK1)mRNA和肿瘤坏死因子受体相关因子6(TRAF6)mRNA的表达,采用Western blot法检测细胞IRAK1和TRAF6的表达。收集细胞上清液,采用ELISA法检测TNF-α、IL-1β和IL-6的浓度。结果与C组比较,LPS组细胞miR-146a表达、IRAK1和TRAF6及其mRNA及表达上调,上清液TNF-α、IL-1β和IL-6的浓度升高(P<0.05);与LPS组比较,LPS-M组细胞miR-146a表达上调,IRAK1和TRAF6及其mRNA表达下调,上清液TNF-α、IL-1β和IL-6的浓度降低,LPS-I组细胞miR-146a表达下调,IRAK1和TRAF6及其mRNA表达上调,上清液TNF-α、IL-1β和IL-6的浓度升高(P<0.05),LPS-NC组上述指标差异无统计学意义(P>0.05)。结论miR-146a是小鼠小胶质细胞炎症反应的内源性保护机制。

Objective To evaluate the role of microRNA(miR)-146a in inflammatory responses in microglial cells of mice.Methods The BV-2 microglial cells of mice were cultured,and the cells at the logarithmic growth phase were divided into 5 groups(n=3 each)using a random number table method:control group(group C),lipopolysaccharide(LPS)group,LPS+miR-146a mimic group(group LPS-M),LPS+miR-146a inhibitor group(group LPS-I)and LPS+negative control group(group LPS-NC).In LPS-M,LPS-I and LPS-NC groups,the miR-146-a mimic RNA,miR-146a inhibitor RNA,and the missense chain of the mimic/inhibitor RNA were transfected into the cells,respectively.After successful transfection,the LPS at the final concentration of 1μg/ml was added in LPS,LPS-M,LPS-I and LPS-NC groups,the equal volume of medium was added in group C,and the cells were incubated for 6 h.The expression of miR-146a,interleukin-1 receptor-associated kinase 1(IRAK1)mRNA and tumor necrosis factor receptor-associated factor 6(TRAF6)mRNA was detected by quantitative real-time polymerase chain reaction,and the expression of IRAK1 and TRAF6 was detected by Western blot.The supernatant was calculated for determination of the concentrations of tumor necrosis factor-alpha(TNF-α),interleukin-1beta(IL-1β)and IL-6 by enzyme-linked immunosorbent assay.Results Compared with group C,the expression of miR-146a and IRAK1 and TRAF6 protein and mRNA was significantly up-regulated,and the concentrations of TNF-α,IL-1βand IL-6 in supernatant were increased in group LPS(P<0.05).Compared with group LPS,the expression of miR-146a was significantly up-regulated,the expression of IRAK1 and TRAF6 protein and mRNA was down-regulated,and the concentrations of TNF-α,IL-1βand IL-6 in supernatant were decreased in group LPS-M,and the expression of miR-146a was significantly down-regulated,the expression of IRAK1 and TRAF6 protein and mRNA was up-regulated,and the concentrations of TNF-α,IL-1βand IL-6 in supernatant were increased in group LPS-I(P<0.05),and no significant change was found in the parameters mentioned above in group LPS-NC(P>0.05).Conclusion miR-146a is an endogenous protective mechanism of inflammatory responses in microglial cells of mice.