微小RNA-146a-5p在新生隐球菌、脂多糖诱导THP-1细胞中表达的对比分析

Expression of microRNA-146a-5p in THP-1 cells following induction by Cryptococcus neoformans versus by lipopolysaccharide

目的为探讨微小RNA-146a-5p(miR-146a-5p)在新生隐球菌所致隐球菌性脑膜炎中的调控作用及机制奠定基础。方法把体外培养的人单核细胞系THP-1细胞分成新生隐球菌刺激组和脂多糖(LPS)刺激组,分别用与细胞数量相同的灭活新生隐球菌(WM148)和浓度为1 mg/L的LPS刺激THP-1细胞,分别于0、3、6、9、12 h收集上清液和细胞。采用酶联免疫吸附试验(ELISA)检测细胞上清液中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6的表达水平,实时荧光定量聚合酶链反应(Real-time PCR)检测细胞miR-146a-5p的表达情况。结果新生隐球菌诱导后,细胞中miR-146a-5p的表达对比对照组(0h)明显升高(P<0.05),3 h达到最高值,随后逐渐下降;上清液中TNF-α的表达在诱导后12 h达到最高值,IL-6的表达各时间点变化不明显。LPS诱导后,miR-146a-5p的表达逐渐升高,12 h达到最高值;上清液中TNF-α的表达3 h后一直处于峰值;IL-6的表达逐渐增加,12 h达到最高值。结论新生隐球菌、LPS诱导THP-1细胞炎性反应后,miR-146a-5p表达随时间延长呈现不同的规律,TNF-α、IL-6的动态变化表现不同的特点。表明miR-146a-5p参与新生隐球菌、LPS诱导THP-1炎性反应存在不同的调控机制。

Objective To explore the underlying mechanism and effect of microRNA-146a-5p（miR-146a-5p）in the cryptococcal meningitis caused by Cryptococcus neoformans.Methods The in vitro cultured THP-1 cells were divided into two groups to subject to induction by Cryptococcus neoformans or by lipopolysaccharide（LPS）.The same amount Cryptococcus neoformans cells（WM148）were added to THP-1 cells to induce expression of miR-146a-5p.Similarly,1 mg/L LPS was added to THP-1cells to stimulate expression of miR-146a-5p.The supernatant and cells were collected separately at 0,3,6,9 and 12 h after incubation.The expression of miR-146a-5p was measured using real-time PCR method.The level of TNF-αand IL-6 in the supernatant was assayed by enzyme-linked immunosorbent assay（ELISA）. Results After induction by Cryptococcus neoformans,the expression of miR-146a-5p increased significantly（P〈0.05）.The peak emerged at 3 h,and then decreased gradually.The level of TNF-αalso increased in the culture supernatant and reached peak at 12 h.The level of IL-6 did not show consistent change at various time points（P〉0.05）.After stimulation by LPS,the expression of miR-146a-5p and level of IL-6 increased gradually and reached peak at 12 h.TNF-αexpression was always at highest level since 3 h after LPS stimulation.Conclusions Following stimulation with Cryptococcus neoformans or LPS,the expression of miR-146a-5p in THP-1 cells showed different patterns over time.The expression levels of TNF-αand IL-6 had different patterns.It suggests that miR-146a-5p is involved in the Cryptococcus neoformans-or LPS-induced THP-1inflammatory reaction via different regulatory mechanisms.