Mitf-m敲除小鼠的基因差异表达分析  

An Analysis of the Gene Expression in Mitf-m Knockout Mice

作　　者：王克双[1,2] 王晓东[1] 任丽丽[1] 陈磊[3] 塞娜[1] 郭维维[1] 杨仕明[1] 

机构地区：[1]中国人民解放军总医院耳鼻咽喉头颈外科,北京100853 [2]南开大学医学院,天津300071 [3]中国农业大学、农业生物技术国家重点实验室,北京100083

出　　处：《中国听力语言康复科学杂志》2016年第1期7-12,共6页Chinese Scientific Journal of Hearing and Speech Rehabilitation

基　　金：国家973计划重大科学研究计划干细胞项目（2012CB967900）;国家973计划重大科学问题导向项目（2011CBA01000）;国家自然科学基金青年项目（81400472）

摘　　要：目的使用RNA-Seq技术分析Miif-m敲除鼠与野生鼠的血管纹转录组，研究Mitf-m基因敲除后的差异表达基因及相关改变的分子机制。方法分别取Miif-m敲除鼠（Mitfmi-△M/mi-△M；MM组）与野生鼠（Miif-m＋/＋；ww组）的血管纹进行总RNA提取；制备cDNA文库，用Illumina HiSeq 2000测序系统行高通量测序。利用软件Tophat2．2．0将cleanreads比对到参考基因组；分别用软件RSeQS-2．3．2和cufflinks来检测测序结果的均一性和基因表达水平。使用edgeR和DESeq程序筛选差异表达基因（differential expressed genes，DEGs）；选择下调DEGs，用KOBAS2．O进行基因本体（geneontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）代谢途径的富集分析。结果MM组与WW组在参考基因组上mapping的cleanreads数分别为42463888和39718542，分别有88．7％和87．4％的reads是唯一映射，说明RNA-Seq结果可靠。DEGs筛查结果表明，相对于WW组，Mftf-m敲除鼠血管纹有45个DEGs，上调表达基因有7个，下调表达基因有38个。GO富集分析发现下调表达基因与黑色素的合成和代谢过程、色素沉着有关，值得关注的是与内向整流钾离子通道Kcnj10和Kcnj13相关。KEGG富集分析显示下调表达基因主要富集于黑色素生成通路。结论RNA-Seq分析拓展了Mftf-m基因调控网，为探究Mitf-m突变所致听力-色素综合征的分子机制及特异性研究Mitf不同转录子的功能奠定了基础。Oblective To compare the stria vascularis transcriptome of the Mitf-m knockout mice and the wild-type mice using RNA sequencing （RNA-Seq）,and to explore the molecular mechanism and functional categories of the differential expressed genes （DEGs）. Methods RNAs were extracted from the stria vascularis of the Mitf-m knockout mice（Mitfmmi-△M/mi-△M;MM group） and the wild-type mice（Mitf-m＋/＋;WW group）, and used to synthesize the cDNA samples, then for RNA-Seq analysis on the Illumina Hiseq 2000 platform. The clean reads were mapped to the reference sequence with program Tophat 2.2.0 and then program RSeQS-2.3.2 and cufflinks were used to evaluate the RNA sequencing results and the gene expression. The program edger and DESeq were applied to identify the DEGs. In order to perform GO term and KEGG enrichment analyses, the down-regulated DEGs were further analyzed using the KOBAS2.0. Results A total of 42463888 and 39718542 clean reads were mapped to the reference sequence in MM group and WW group. For screening DEGs, 88.7% and 87.4% of the reads were uniquely mapped to the reference sequence, demonstrating the high confidence of the RNA-Seq results. Among the 45 DEGs, 7 and 38 genes were detected to be up-and down-regulated, respectively. GO enrichment analysis indicated that the down-regulated genes were mainly enriched in pigmentation and melanin biosynthesis and metabolic process. DEGs were involved with the inward-rectifier potassium ion channel（Kcnjl0 and Kcnjl3）activity. KEGG enrichment analysis showed that the down-regulated DEGs were mainly enriched in melanogenesis pathways. Conclusion The data provide a large amount of useful information about mRNAs that are related in Mitf-m related pathways. The results will help to further understand the molecular function categories and pathways of Mitf.

关 键 词：Mitf-m基因 血管纹 RNA-Seq技术 差异表达基因 

分 类 号：R73[医药卫生—肿瘤]