The role of miR-17-92 in the miRegulatory landscape of Ewing Sarcoma (RNA-Seq)

MicroRNAs serve to fine-tune gene expression and play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a tissue still poses a significant problem since the presence of a seed sequence in the 3´UTR of an mRNA and its expression modulation upon ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing Sarcoma. miR-17-92, one of the most potent oncogenic miRNAs, was recently reported to be the top EWS-FLI1 activated miRNA. Using a combination of AGO2 pull-down experiments by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) and of RNAseq upon miRNA depletion by ectopic sponge expression, we aimed to identify the targetome of miR-17-92 in Ewing sarcoma. Intersecting both datasets we found an enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3´UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Strikingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signalling. Taken together, our findings shed light on the complex miRegulatory landscape of Ewing Sarcoma pointing miR-17-92 as a key node connected to TGFB/BMP pathway Overall design: mRNA profiles of a Ewings Sarcoma cellline (clone of A673 with inducible sh EWS-FLI1 knockdown) treated with microRNA sponges and controls

微小RNA（microRNAs, miRNAs）可精细调控基因表达，在组织特异性基因网络中发挥关键调控作用。在组织中鉴定并验证miRNA靶基因仍是一项重大挑战：mRNA的3'非翻译区（3'UTR）若存在种子序列，以及异位表达miRNA后其表达发生调控，这两种情况均无法可靠预测生理条件下的真实调控效应。
嵌合癌蛋白EWS-FLI1是尤因肉瘤（Ewing Sarcoma）的核心致病驱动因子。而miR-17-92作为致癌性最强的miRNA之一，近期被报道为受EWS-FLI1激活程度最高的miRNA。
本研究结合光活化核糖核苷增强交联免疫沉淀（Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation, PAR-CLIP）技术的AGO2下拉实验，以及通过异位表达miRNA海绵敲低miRNA后的RNA测序（RNAseq）数据，旨在鉴定尤因肉瘤中miR-17-92的靶标组（targetome）。
对两组数据集进行交集分析后发现，经miR-17-92特异性海绵处理的样本中，miR-17-92家族成员的PAR-CLIP结合位点在上调基因的3'UTR中显著富集。尤为关键的是，其中约四分之一的基因注释至TGFB/BMP通路，且大多数定位于SMAD信号通路的下游。
综上，本研究结果揭示了尤因肉瘤复杂的miRNA调控图谱，明确miR-17-92是连接TGFB/BMP通路的核心节点。
整体实验设计：对携带可诱导敲低EWS-FLI1短发夹RNA的A673细胞克隆（尤因肉瘤细胞系）分别用miRNA海绵及对照处理后，获取其mRNA表达谱。