Small RNA-sequencing reveals the involvement of microRNA-132 in benzo(a)pyrene-induced toxicity in primary human blood cells

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental contaminants, triggering deleterious effects such as carcinogenicity and immunosuppression and peripheral blood mononuclear cells (PBMCs) are among the main cell types targeted by these pollutants. In the present study, we sought to identify the expression profiles and function of miRNAs, gene regulators involved in major cellular processes and recently linked to environmental pollutants, in PBMC-exposed to the prototypical PAH, benzo[a]pyrene (B[a]P). Using small RNA deep sequencing, we identified several B[a]P-responsive miRNAs. Bioinformatics analyses showed that their predicted targets could modulate biological processes relevant to cell death and survival. Further studies of the most highly induced miRNA, miR-132, showed that its up-regulation by B[a]P was time- and dose-dependent and required aryl hydrocarbon receptor (AhR) activation. By evaluating the role of miR-132 in B[a]P-induced cell death, we propose a mechanism linking B[a]P-induced miR-132 expression and CYP1A1 and CYP1B1 mRNA levels, which could contribute to the apoptotic response of PBMCs. Altogether, this study increases our understanding of the roles of miRNAs induced by B[a]P and provides the basis for further investigations into the mechanisms of gene expression regulation by PAHs. Overall design: 12 independent PBMC cultures each grown in untreated (DMSO) and 2 µM B[a]P-treated conditions for 48 h, were combined in 3 equal pools (with 4 separate blood donors per pool) for each condition. They were then used to isolate 5-15 ng of the small RNA fraction and processed using Qiaseq miRNA library prep kit

多环芳烃（Polycyclic Aromatic Hydrocarbons, PAHs）是一类广泛分布的环境污染物，可引发致癌性、免疫抑制等有害效应；外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）是这类污染物的主要靶细胞类型之一。本研究旨在探究暴露于典型多环芳烃苯并[a]芘（Benzo[a]Pyrene, B[a]P）的外周血单个核细胞中，微小RNA（microRNAs, miRNAs）——一类参与核心细胞进程、且近期被发现与环境污染物相关的基因调控因子——的表达谱与功能。本研究采用小RNA深度测序技术，筛选出若干受B[a]P调控的miRNAs。生物信息学分析显示，这些miRNA的预测靶基因可调控与细胞死亡及存活相关的生物学过程。针对诱导幅度最高的miRNA——miR-132的进一步研究表明，B[a]P对其的上调作用呈时间与剂量依赖性，且需要芳烃受体（Aryl Hydrocarbon Receptor, AhR）的激活。本研究通过探究miR-132在B[a]P诱导的细胞死亡中的作用，提出了一条关联B[a]P诱导的miR-132表达与CYP1A1、CYP1B1 mRNA水平的调控机制，该机制或可参与PBMCs的凋亡应答过程。综上，本研究加深了我们对B[a]P诱导的miRNAs功能的理解，并为进一步探究多环芳烃调控基因表达的机制提供了研究基础。实验设计：共设置12组独立的PBMC培养体系，分别于未处理（二甲基亚砜，DMSO）及2 μM B[a]P处理条件下培养48小时；随后将每个处理组的样本按4名不同供血者的样本混合为1份混合样，每组共3份混合样。之后从每份混合样中提取5~15 ng的小RNA组分，并采用Qiaseq miRNA文库制备试剂盒完成文库构建。