miR-146a在人牙髓细胞中的表达及其作用研究

目的 检测miR-146a在正常及脂多糖（LPS）刺激的人牙髓细胞（hDPC）中的表达,探讨miR-146a对hDPC分泌炎性因子的影响及其机制.方法 （1）实时荧光定量聚合酶链反应（PCR）方法测定正常及LPS刺激的hDPC miR-146a的表达水平.（2） Lipofectamine 2000脂质体分别转染化学合成的miR-146a类似物（miR-146a mimics）、miR-146a抑制剂（miR-146a inhibitor）及其相应无关序列小RNA对照,转染24 h后1μg/ml LPS刺激hDPC,4h后实时荧光定量PCR检测白细胞介素6（IL-6）、IL-8 mRNA的表达,酶联免疫吸附试验（ELISA）检测细胞上清IL-6、IL-8的分泌;Western blot 法检测miR-146a下游靶标白细胞介素1受体相关激酶1（IRAK1）及肿瘤坏死因子受体相关因子6（TRAF6）的蛋白表达水平.两组比较采用独立样本t检验,多组比较采用方差分析.结果 （1）经LPS刺激的hDPC miR-146a表达水平高于正常hDPC（12 h：t=8.488,P=0.014;24 h：t=39.661,P＜0.001）.（2）转染miR-146a mimics的hDPC在1μg/mlLPS刺激下产生炎性因子IL-6、IL-8的水平低于阴性对照,差异有统计学意义（IL-6 PCR：P＜0.001,IL-6 ELISA：P＜0.001;IL-8 PCR：P＜0.001,IL-8ELISA：P=0.011）;过表达miR-146a后IRAK1及TRAF6蛋白水平明显下调（IRAK1：P=0.002; TRAF6：P＜ 0.001）.结论 miR-146a在LPS刺激的hDPC中表达上调,转染miR-146a可下调其靶基因IRAK1及TRAF6表达并降低IL-6、IL-8分泌,推测miR-146a参与牙hDPC的炎症反应调控.

A study on the expression and function of miR-146a in human dental pulp cells

Objective To investigate the expression of miR-146a in normal human dental pulp cells （hDPCs） and hDPCs stimulated by lipopolysaccharide （LPS）,and explore the possible role of miR-146a in pulpitis.Methods The expression levels of miR-146a in normal human dental pulp cells and hDPCs stimulated by LPS were detected by real-time PCR.Moreover,miR-146a mimics and miR-146a inhibitor,as well as their counterpart negative controls were transfected into cultured hDPCs by Lipofectamine 2000 for 24 h,followed by 1 μg/ml LPS stimulation for 4 h.MRNA of IL-6 and IL-8 were tested by real-time quantitative PCR,and IL-6 and IL-8 levels in the culture supernatant were detected by ELISA.Moreover,IRAK1 and TRAF6 which previously identified as actual target of miR-146a were examined by Western blot.Comparisons between groups were performed with t test or one-way ANOVA analysis.Results It was shown that miR-146a was significantly up-regulated in LPS treated hDPCs compared with normal hDPCs （12 h：t=8.488,P =0.014 ; 24 h：t=39.661,P ＜ 0.01）.Up-regulation of miR-146a could decrease secretion of IL-6 and IL-8 both in mRNA and supernatant levels （IL-6 PCR：P＜0.001,IL-6 ELISA：P＜0.001; IL-8 PCR：P＜0.001,IL-8 ELISA：P=0.011）.The expression of IRAK1 and TRAF6 of hDPCs transfected with miR-146a mimics was obviously down-regulated compared to those transfected with mimics control （IRAK1：P=0.002; TRAF6：P ＜ 0.001）.Conclusions MiR-146a is up-regulated in hDPCs induced by LPS.Up-regulation of miR-146a could not only down-regulate IRAK1 and TRAF6 expression,but also decrease IL-6 and IL-8 Ievels.This indicates miR-146a may be involved in the regulation of pulpitis.