MicroRNAs in differentiation of muscle

描述（由申请人提供）：MicroRNAs作为基因组的暗物质进入分子生物学。它们是在哺乳动物细胞中产生的20-22个碱基长的rna，直到最近十年才被发现。直到现在，我们才开始认识到它们在哺乳动物细胞中调节基因表达方面所起的关键作用。C2C12成肌细胞向肌管的分化是成人骨骼肌卫星细胞向肌管分化的典范。我们假设在肌肉分化过程中诱导的microrna对诱导细胞静止和促进肌肉分化很重要。本项目的目的是表征骨骼肌细胞的microRNAome，目的是检测参与C2C12成肌细胞和原代卫星细胞分化的microrna。我们已经确定了在分化过程中诱导的五个microrna，并计划识别更多行为相似的microrna。特异性microRNAs水平的选择性去调控（增加和减少）将决定microRNAs如何影响细胞静止和肌肉细胞的分化。初步结果表明，参与DNA复制和细胞周期进程的两个关键基因是microrna的靶标。我们将在成肌细胞和非肌肉细胞中确定这些和其他基因的microrna下调在细胞静止建立中的作用。最后，microrna的直接靶标将通过微阵列杂交和生物信息学双管齐下的方法确定。对建立细胞静止重要的假定靶点将进行测试，以确定它们是否确实在分化过程中受到microrna的抑制，以及这种抑制如何促进细胞静止。

DESCRIPTION (provided by applicant): MicroRNAs have burst into molecular biology as the dark matter of the genome. They are 20-22 base long RNAs produced in mammalian cells that have gone undetected till the last decade. Only now are we beginning to realize the critical role they play in regulation of gene expression in mammalian cells. The differentiation of C2C12 myoblasts into myotubes is paradigmatic of the differentiation of satellite cells into myotubes in adult skeletal muscle. We hypothesize that microRNAs induced during muscle differentiation are important for inducing cell quiescence and promoting myogenic differentiation. The goal of this project is to characterize the microRNAome of skeletal muscle cells with the intent of detecting microRNAs involved in differentiation of C2C12 myoblasts and primary satellite cells. We have identified five microRNAs that are induced during the differentiation process and plan to identify any more that behave similarly. Selective de-regulation of the levels of specific microRNAs (both increase and decrease) will determine how the microRNAs affect cell quiescence and differentiation of the muscle cells. Preliminary results reveal that two key genes involved in DNA replication and cell cycle progression are targets of the microRNAs. We will determine the role of down regulation of these and other genes by the microRNAs in establishment of cell-quiescence in both myoblasts and non-muscle cells. Finally the direct targets of the microRNAs will be ascertained by a two-pronged approach using microarray hybridizations and bioinformatics. Putative targets important for establishment of cell quiescence will be tested to ascertain whether they are indeed repressed by microRNAs during differentiation and how such repression contributes to cell quiescence.