微小RNA-146a在新生隐球菌、格特隐球菌诱导THP-1细胞炎性反应中的机制研究

Mechanistic study on the role of microRNA-146a in THP-1 cells-associated inflammatory response induced by Cryptococcus neoformans and Cryptococcus gattii

目的观察并分析微小RNA-146a(miR-146a)在新生隐球菌(标准株WM148)、格特隐球菌(标准株R265)刺激人单核巨噬细胞系(THP-1细胞)中表达的差异,探讨miR-146a在隐球菌所致隐球菌脑膜炎中的调控作用及机制。方法体外培养的人单核巨噬细胞系THP-1细胞分成新生隐球菌诱导组、格特隐球菌诱导组,按灭活菌数∶THP-1=5∶1的比例与THP-1细胞共孵育0、3、6、9和12 h后离心收集上清液和细胞沉淀。采用实时荧光定量PCR(q RT-PCR)技术检测各组细胞miR-146a的表达情况,酶联免疫吸附试验(ELISA)法检测各组上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的释放量。结果新生隐球菌诱导组细胞中miR-146a表达量与0 h相比明显升高(P<0.01),且在3 h达到峰值,随后呈下降趋势;上清液中TNF-α的表达量在诱导后12 h达到最高值,IL-6的表达各时间点没有明显变化。格特隐球菌诱导后,miR-146a的表达逐渐升高,12 h达到最高值,但3 h、6 h点变化与0 h比较差异并无统计学意义;上清液中TNF-α的表达12 h到达峰值;IL-6的表达逐渐增加,12 h达到最高值,变化无统计学意义。结论新生隐球菌、格特隐球菌刺激THP-1细胞炎性反应后,miR-146a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。研究表明新生隐球菌、格特隐球菌诱导THP-1炎性反应可能存在不同的调控机制。

Objective To analyze the differential expression of microRNA-146a （miR-146a） in monocyte-macrophage cell line （THP-1 cells） after induction by Cryptococcus neoformans （C. neoformans, reference strain WM148） or Cryptococcus gattii （C. gattii, reference strain R265）, and investigate the mechanism of miR-146a in regulating the inflammatory response of cryptococcal meningitis. Methods The cultured THP-1 cells were divided into two groups to be induced by C. neoformans or C. gattii, respectively. THP-1 cells were induced with inactivated WN148 （or R265） strains at multiplicity of infection （MOI） of 5 in all experiments. The supernatant and the cell pellet were collected separately after incubation. The expression of miR-146a was measured by real-time quantitative PCR （qRT-PCR） techniqueThe levels of TNF-α and IL-6 release were assayed by ELISA. Results The expression of miR-146a increased significantly in the C. neoformans induction group compared to 0 h. It reached peak at 3 h （P〈0.01）, and then declined gradually. The level of TNF-α increased in supernatant and reached peak at 12 h. The expression of IL-6 did not change significantly at each time point. The expression of miR-146a and TNF-α increased gradually and reached peak at 12 h in the C. gattii induction group （P 〈0.01）, but the change did not reach statistical significance at 3 h, 6 h time points. The expression of IL-6 gradually increased, and reached peak at 12 h time point. Conelnsions Following stimulation with C. neoformans or C. gattii, the expression of miR-146a in THP-1 cells showed different patterns over time. The expression levels of TNF-α and IL-6 showed different patterns. These findings suggest that there may be different regulatory mechanisms in the THP-1 cells-associated inflammatory response after stimulation by inactivated C. neoformans and C. gattii strains.