Transcription profiling of mouse hearts from wild type and miRNA-1-2 knockout mutants at postnatal day 10 to provide insight into the role of miRNA-1-2 in cardiac development

Microarray was done on heart tissue from ko and wt; MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the dosage of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals is unknown, and reliable prediction of mRNA targets remains problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets. Experiment Overall Design: Heart tissues from 3 wild type and 3 miR-1-2 knockout mice at postnatal days 10 were used and total RNA was extracted by Trizol. Expression level was compared between wild type and miR-1-2 knockout mice. The affy package from R/Bioconductor was used to generate RMA values.本研究对敲除（knockout, KO）与野生型（wild type, WT）小鼠的心脏组织开展了基因芯片（microarray）检测。微小RNA（microRNAs, miRNAs）是由基因组编码的小型RNA分子，可通过信使RNA（messenger RNA, mRNA）转录本调控生物体蛋白质的合成剂量。目前，哺乳动物体内特定miRNAs的在体生理需求尚不明确，且可靠预测mRNA靶标的技术仍存在诸多难题。本研究证实，小鼠心脏中的miRNA生物发生过程对心脏发育至关重要。此外，靶向敲除肌肉特异性miRNA——miR-1-2后，研究发现其在心脏中具有多种功能，包括调控心脏形态发生、电传导以及细胞周期调控。通过分析miR-1-2敲除后表达上调的mRNA中miR-1的互补序列，我们发现miR-1种子序列匹配位点显著富集，且潜在的miR-1结合位点倾向于位于物理可及的RNA区域。上述研究结果表明，miRNA剂量的细微改变即可对哺乳动物产生深远影响，并证实了哺乳动物功能丧失模型在揭示生理相关miRNA靶标方面的应用价值。实验整体设计：选取出生后第10天的3只野生型小鼠与3只miR-1-2敲除小鼠的心脏组织，采用Trizol法提取总RNA，对比两组小鼠的基因表达水平。使用R/Bioconductor的affy软件包生成RMA标准化值。