长链非编码RNA AABR07008568.1在PM2.5致NR8383细胞炎症反应中的作用

Role of long non-coding RNA AABR07008568.1 in inflammation of NR8383 cells induced by PM2.5

目的探讨长链非编码RNA(lncRNA)AABR07008568.1在大气PM2.5诱导大鼠肺泡巨噬细胞(NR8383)炎症反应中的作用及其分子机制。方法采集广州地区现场实时PM2.5样品制成混悬液用于后续实验的染毒,用0、200μg/ml PM2.5染毒液暴露NR8383细胞24 h后,采用lncRNA高通量测序并结合生物信息学分析方法筛选出有差异表达的lncRNAs,使用实时荧光定量PCR分析法(RT-qPCR)验证测序结果。运用胞浆胞核亚细胞定位试剂盒检测AABR07008568.1在胞浆胞核的分布情况。采用RNA干扰(RNAi)技术敲低NR8383细胞中AABR07008568.1的表达水平,以RT-qPCR检测RNA干扰联合200μg/ml PM2.5染毒液暴露NR8383细胞后IL-6的表达水平。用NFκB特异性抑制剂(BAY11-7082,10μmol/L)处理细胞后检测AABR07008568.1的表达水平。结果高通量测序和RT-qPCR结果显示,随着PM2.5暴露浓度的增加,AABR07008568.1表达量增高(P<0.05)。通过干扰AABR07008568.1后发现siRNA1的转染效率最高,达到约60%(P<0.05)。敲低AABR07008568.1且联合PM2.5暴露后,与阴性对照(NC)组相比,siRNA1组中IL-6的表达水平降低,差异具有统计学意义(P<0.05);NC+PM2.5组中的IL-6表达上调,差异具有统计学意义(P<0.05)。当使用NFκB特异性抑制剂阻断NFκB通路后,AABR07008568.1表达量下降(P<0.05)。结论AABR07008568.1参与了NFκB通路的激活,但两者之间具体的调控机制尚需更深层次的探索。

Objective To understand the role and molecular mechanism of long non-coding RNA AABR07008568.1 in inflammatory response in NR8383 cell line exposed to PM2.5.Methods The real-time PM2.5 samples were collected from Guangzhou and NR8383 cells were treated with PM2.5 at the doses of 0μg/ml and 200μg/ml for 24 hours,the significantly different lncRNAs were screened out by long-chain non-coding(lnc)RNA sequencing and bioinformatics analysis.Non-coding RNA sequencing results were validated by RT-qPCR.Subcellular localization of gene was performed using a cytoplasmic nuclear isolation kit.The expression of AABR07008568.1 were knocked down through siRNA interfering,and IL-6 expression level was detected by RT-qPCR after siRNA interfering combined 200μg/ml PM2.5 exposure.BAY11-7082(10μmol/L),a specific blocker of NF-kappa B,was used to detect the change of AABR07008568.1 after blocking NF-kappa B pathway.Results The results of long-chain non-coding(lnc)RNA sequencing and RT-qPCR indicated that with the increasing concentration of PM2.5,it induced up-regulation of AABR07008568.1 in NR8383(P<0.05).siRNA1 was the best siRNA fragment,with the highest transfection efficiency about 60%.After interference with AABR07008568.1 combined PM2.5 exposure,compared to negative control(NC)group,the IL-6 level of siRNA1 group significantly decreased(P<0.05),the IL-6 level of NC+PM2.5 group significantly increased(P<0.05).The level of AABR07008568.1 was significantly decreased after blocking NF-kappa B pathway(P<0.05).Conclusion AABR07008568.1 is involved in activating the NF-kappa B pathway,but the specific regulation mechanism between them still need further exploration.