Overexpression of Mir-197 und Mir-769 in human iPSCs

MicroRNAs (miRNAs) are post-transcriptional regulators that are involved in many essential biological processes. Recently, targeted searches for human miRNA orthologs revealed that 15 miRNA families are lost in the Eumuroidea lineage, including the model organisms mouse and rat, but not in other mammals. We here characterize this loss in more detail and investigate potential consequences for the Eumuroidean regulatory network. We confirm that the miRNA loss was not compensated by other miRNAs in rat or mouse, and we identify a significant overlap in the function of their target genes. We heterologously express two human miRNAs, Mir-197 and Mir-769, in mouse inducible pluripotent stem cells (iPSCs) and compare effects on the transcriptome to those of induced miRNA levels in human iPSCs. This reveals overlapping sets of down-regulated mRNAs in the two species, likely representing remnants of an ancient miRNA-dependent regulatory network that predates the diversification of the human and mouse lineages. The consequent elimination of established miRNA regulatory connections on the Eumuroidean lineage is accompanied by the lineage-specific loss of 37 transcription factors. Our results indicate that mouse and rat have modified their regulatory network of gene expression on transcriptional and post-transcriptional level compared to other vertebrate model organisms. This regulatory shift likely adds noise to studies of gene expression that, for example, use murine cells to study human diseases. Overall design: Differential gene expression analysis upon overexpression for 48h of either miRNA Precursor hsa-miR-197-3p or miRNA Precursor hsa-miR-769-5p in human iPSCs compared to miRNA Precursor Negative Control

微小RNA（MicroRNAs，miRNAs）是一类参与诸多核心生物学过程的转录后调控因子。近年来，针对人类miRNA直系同源物的靶向筛选研究显示，共有15个miRNA家族在真鼠总科（Eumuroidea）谱系中发生了丢失，该谱系包含模式生物小鼠与大鼠，但其他哺乳动物并未出现此类丢失情况。本研究对该miRNA丢失事件开展了更为细致的表征，并探究了其对真鼠类基因调控网络的潜在影响。
我们证实，大鼠与小鼠体内的其他miRNA并未对此次miRNA丢失产生补偿作用，同时发现二者靶基因的功能存在显著重叠。我们将两种人类miRNA——Mir-197与Mir-769——在小鼠诱导多能干细胞（induced pluripotent stem cells，iPSCs）中进行异源表达，并将其对转录组的影响与人类iPSCs中诱导表达的上述miRNA所引发的转录组变化进行对比。
研究结果显示，两个物种中均出现了下调mRNA集合的重叠现象，这大概率代表了在人类与小鼠谱系分化之前就已存在的、依赖miRNA的古老调控网络的遗迹。在真鼠总科谱系中，已确立的miRNA调控连接随之被消除，同时伴随37个转录因子的谱系特异性丢失。
我们的研究结果表明，相较于其他脊椎动物模式生物，小鼠和大鼠在转录及转录后水平上均对自身基因表达调控网络进行了改造。这种调控转变可能会给相关基因表达研究带来干扰——例如那些利用小鼠细胞开展人类疾病研究的工作。
整体实验设计：在人类iPSCs中过表达miRNA前体hsa-miR-197-3p或miRNA前体hsa-miR-769-5p，并以miRNA前体阴性对照作为参照，培养48小时后进行差异基因表达分析。