miR-155 targets Caspase-3 mRNA in activated macrophages

To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan^TM we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. <i>In vitro</i> translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation.

为维持固有免疫应答中活化巨噬细胞的功能，需对其存活时长进行精准调控。细菌脂多糖（lipopolysaccharides, LPS）通过Toll样受体4（toll-like receptor 4, TLR4）识别后，可激活下游信号通路，最终诱导细胞因子基因与抗凋亡基因的表达。微小RNA（MicroRNAs, miRNAs）现已被证实为重要的炎症应答调控因子，但目前关于其对细胞凋亡的功能影响的研究仍较为匮乏。为筛选经LPS刺激后差异表达的miRNAs，本研究通过深度测序分析了未处理与经LPS活化的小鼠巨噬细胞的cDNA文库，并通过Northern印迹（Northern blotting）与实时定量PCR（qPCR）验证了差异表达的miRNA。本研究借助TargetScan^TM软件，将编码凋亡关键调控因子的半胱天冬酶-3（CASPASE-3, CASP-3）mRNA鉴定为LPS诱导的miR-155的潜在靶标。经LPS刺激的原代巨噬细胞活化实验显示，TLR4介导了miR-155表达的上调与CASP-3 mRNA水平的降低。在经LPS活化的巨噬细胞中，内源性CASP-3 mRNA与剪切活化的CASP-3蛋白水平均出现下降。通过Argonaute 2（AGO2）免疫沉淀实验，证实miR-155与CASP-3 mRNA可富集于miRNA诱导沉默复合物（miRISC）中。重要的是，特异性miRNA拮抗剂（antagomir）转染可有效降低成熟miR-155的水平，并使活化巨噬细胞中的CASP-3 mRNA水平显著升高。体外（<i>In vitro</i>）翻译实验证实，CASP-3 mRNA 3'非翻译区（3'UTR）中的靶位点可介导miR-155依赖的荧光素酶（Luciferase）报告mRNA降解。值得注意的是，对转染了antagomir-155并经LPS预处理、再予以星形孢菌素（staurosporine, SSP）处理的巨噬细胞进行膜联蛋白V（Annexin V）染色结果显示，LPS诱导的miR-155可通过下调CASP-3 mRNA水平抑制细胞凋亡。综上，本研究证实，在经LPS活化的RAW 264.7巨噬细胞中，miR-155介导的CASP-3 mRNA降解可抑制细胞凋亡，这是维持巨噬细胞在炎症应答中核心功能的必要前提。