Identification of dysregulated genes after deletion of miR-1-1 and miR-1-2

Purpose: The goals of this study are to identify dysregulated mRNAs in the heart after deletion of miR-1-1 and miR-1-2. Methods: Total RNAs were extracted from hearts at embryonic E15.5 (E15.5) or postnatal 2.5 days (P2.5) of miR-1s dHET and miR-1s dKO mice. mRNAs were purified using a poly-A selection approach and sequenced using Illumina HiSeq 2000. The sequence reads were aligned to the mouse reference genome (NCBI Build 37/mm9) using TopHat program (Bowtie algorithm). Transcript assembles and identification of differentially expressed genes were achieved using Cufflinks package. To account for expression bias due to transcript length, each sample transcript expression was normalized by using cufflinks algorithm with a FDR of 0.05. Results: Using 1.5-fold change as a cutoff, 997 and 653 transcripts were found to be upregulated and downregulated, respectively, in miR-1s dKO heart at P2.5. 423 transcripts were found to be upregulated and 653 were down-regulated in miR-1s dKO heart at E15.5. Many upregulated genes are directly involved in a fetal gene program. Conclusions: miR-1 directly represses a fetal gene program. We performed RNA deep-seq using postnatal 2.5 days (P2.5) heart from miR-1-1 and miR-1-2 double knockout mice and compared it to that of littermate control.研究目的：本研究旨在鉴定微小RNA-1-1（microRNA-1-1，miR-1-1）与微小RNA-1-2（microRNA-1-2，miR-1-2）敲除后小鼠心脏内失调的信使RNA（messenger RNA，mRNA）。 实验方法：分别提取miR-1双杂合（miR-1s dHET）与miR-1双敲除（miR-1s dKO）小鼠胚胎期第15.5天（E15.5）及出生后第2.5天（P2.5）的心脏总RNA。采用poly(A)富集法纯化mRNA，并使用Illumina HiSeq 2000测序平台进行测序。利用TopHat程序（基于Bowtie算法）将测序读段比对至小鼠参考基因组（NCBI Build 37/mm9）。通过Cufflinks软件包完成转录本组装与差异表达基因鉴定。为校正转录本长度带来的表达偏差，采用Cufflinks算法对各样本的转录本表达量进行标准化处理，设定错误发现率（False Discovery Rate, FDR）为0.05。 实验结果：以1.5倍表达变化作为筛选阈值，在P2.5期的miR-1s dKO小鼠心脏中，分别鉴定得到997个上调转录本与653个下调转录本；在E15.5期的miR-1s dKO小鼠心脏中，鉴定得到423个上调转录本与653个下调转录本。其中大量上调基因直接参与胎儿基因程序（fetal gene program）。 研究结论：miR-1可直接抑制胎儿基因程序。本研究对出生后第2.5天（P2.5）的miR-1-1与miR-1-2双敲除小鼠心脏组织进行了RNA深度测序（RNA deep-seq），并将其与同窝对照样本进行了比对分析。