长非编码RNA Rian/Irm在小鼠骨骼肌发育中的功能研究

Long non-coding RNA ( long non-coding RNA, lncRNA )have the functions in epigenetic transcriptional and posttranscriptional regulation, such as level of regulation of gene expression. Recent studies show that, long non-coding RNA can regulate the proliferation and differentiation of skeletal muscle cells, suggesting that ncRNA regulate on skeletal muscle development. Rian is a maternal expression of long non-coding RNA gene, and is located on mouse chromosome 12 Dlk1-Dio3 domain, the gene encoding a plurality of miRNAs and snoRNAs. Our preliminary work had studied three transcription of the Rian gene-Meg8,Irm and AB063319 -their tissue expression pattern, and positioning in the nucleus, the results show that in the blastocyst stage expression started and continued after birth, and it expressed in a variety of skeletal muscle tissue. Rian gene in skeletal muscle had a wider role; we detected the DMR regulation sequence of GTl2 in Leydig cell lines,it existed the differential methylation, suggesting that it may result in Rian gene transcripts differentially expressed reasons. Therefore, we will study in mouse myoblasts and embryo by over expression and interference/knockout to change the expression of Rian gene transcription Irm, through detecting high-throughput analysis of the results, using molecular biology techniques to verification the Irm role in skeletal muscle and functional relationship with the Dlk1-Dio3 domain, to clarify lncRNA role in regulating skeletal muscle development and function analysis of ncRNA and genomic imprinting studies provide a theoretical basis.

长非编码RNA（long non-coding RNA, lncRNA）在表观遗传、转录及转录后调控等多种层面上调控基因的表达。近年研究表明，lncRNA能够调节骨骼肌细胞的增殖与分化，提示lncRNA对骨骼肌的发育具有调控作用。Rian作为一个母源表达的lncRNA基因，编码多个miRNAs和snoRNAs。课题前期工作发现，该基因的三个转录本Meg8、Irm 和AB063319 在多种胚胎组织中持续表达，其中Irm在骨骼肌中的表达尤其显著，提示其在胚胎骨骼肌发育中具有特殊作用。本研究将在小鼠成肌细胞及胚胎中改变和敲除Irm的表达，在细胞与个体模型中进行表达谱高通量检测，全基因组甲基化分析，并通过形态学观察和分子生物学手段解析和揭示Irm在骨骼肌发育中的作用及与Dlk1-Dio3 domain基因表达的相互关系，为阐明lncRNA对骨骼肌发育的调控作用及ncRNA的功能解析提供理论依据。

长非编码RNA（long non-coding RNA, lncRNA）在表观遗传、转录及转录后调控等多种层面上调控基因的表达。近年研究表明，lncRNA能够调节骨骼肌细胞的增殖与分化，提示lncRNA对骨骼肌的发育具有调控作用。Rian作为一个母源表达的lncRNA基因，编码多个miRNAs和snoRNAs。课题前期工作发现，该基因的三个转录本Meg8、Irm 和AB063319 在多种胚胎组织中持续表达，其中Irm在骨骼肌中的表达尤其显著，提示其在胚胎骨骼肌发育中具有特殊作用。本研究结果显示：在整个胚胎发育阶段，Rian在胚胎E8.5~E19.5时期呈现持续表达状态，并且从E8.5到E18.5呈现逐渐升高的趋势，但是在E19.5其表达量下降到E18.5的25%左右，提示Rian功能可能主要呈现在胚胎发育过程；定量RT-PCR分析Rian的三个转录本在小鼠E13.5、15.5、17.5、19.5胚胎舌、心脏和四肢肌肉中的表达情况，观察到：在骨骼肌和舌中随发育的进行Irm的表达水平逐渐提高，提示其在胚胎横纹肌组织的发育中具有一定的调控作用；Rian的三个转录本在C2C12细胞分化过程中的表达呈现不同，Irm 和AB063319呈现升高的趋势，而Meg8不表达；为了在小鼠成肌细胞系中改变Irm的表达，设计了Irm的过表达载体和干扰shRNA，转染C2C12细胞系，将通过形态学观察、芯片检测及分子生物学手段来解析和揭示Irm在骨骼肌发育中的作用，其结果将为阐明lncRNA对骨骼肌分化、发育的调控作用及ncRNA的功能解析提供理论依据。