Genomics and Computational Biology Graduate Group, 1420 Blockley Hall, 423 Guardian Drive, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
BMC Biol. 2013 Feb 28;11:19. doi: 10.1186/1741-7007-11-19.
The nuclear factor-KappaB (NF-κB) pathway is conserved from fruit flies to humans and is a key mediator of inflammatory signaling. Aberrant regulation of NF-κB is associated with several disorders including autoimmune disease, chronic inflammation, and cancer, making the NF-κB pathway an attractive therapeutic target. Many regulatory components of the NF-κB pathway have been identified, including microRNAs (miRNAs). miRNAs are small non-coding RNAs and are common components of signal transduction pathways. Here we present a cell-based functional genomics screen to systematically identify miRNAs that regulate NF-κB signaling.
We screened a library of miRNA mimics using a NF-κB reporter cell line in the presence and absence of tumor necrosis factor (+/- TNF). There were 9 and 15 hits in the -TNF and +TNF screens, respectively. We identified putative functional targets of these hits by integrating computational predictions with NF-κB modulators identified in a previous genome-wide cDNA screen. miR-517a and miR-517c were the top hits, activating the reporter 86- and 126-fold, respectively. Consistent with these results, miR-517a/c induced the expression of endogenous NF-κB targets and promoted the nuclear localization of p65 and the degradation of IκB. We identified TNFAIP3 interacting protein1 (TNIP1) as a target and characterized a functional SNP in the miR-517a/c binding site. Lastly, miR-517a/c induced apoptosis in vitro, which was phenocopied by knockdown of TNIP1.
Our study suggests that miRNAs are common components of NF-κB signaling and miR-517a/c may play an important role in linking NF-κB signaling with cell survival through TNIP1.
核因子-κB（NF-κB）途径从果蝇到人都是保守的，是炎症信号转导的关键介质。NF-κB 的异常调节与包括自身免疫疾病、慢性炎症和癌症在内的几种疾病有关，这使得 NF-κB 途径成为一个有吸引力的治疗靶点。NF-κB 途径的许多调节成分已经被确定，包括 microRNAs（miRNAs）。miRNAs 是小的非编码 RNA，是信号转导途径的常见组成部分。在这里，我们提出了一种基于细胞的功能基因组筛选方法，用于系统地鉴定调节 NF-κB 信号的 miRNAs。
我们在存在和不存在肿瘤坏死因子（+/-TNF）的情况下，使用 NF-κB 报告细胞系筛选了 miRNA 模拟物文库。在-TNF 和 +TNF 筛选中分别有 9 和 15 个命中。我们通过将计算预测与之前全基因组 cDNA 筛选中鉴定的 NF-κB 调节剂相结合，鉴定了这些命中的潜在功能靶点。miR-517a 和 miR-517c 是排名最高的命中，分别激活报告基因 86 倍和 126 倍。与这些结果一致，miR-517a/c 诱导内源性 NF-κB 靶基因的表达，并促进 p65 的核定位和 IκB 的降解。我们鉴定了 TNFAIP3 相互作用蛋白 1（TNIP1）作为靶标，并对 miR-517a/c 结合位点的功能性 SNP 进行了特征描述。最后，miR-517a/c 在体外诱导细胞凋亡，这可被 TNIP1 的敲低所模拟。
我们的研究表明，miRNAs 是 NF-κB 信号的常见组成部分，miR-517a/c 可能通过 TNIP1 在将 NF-κB 信号与细胞存活联系起来方面发挥重要作用。
