基于转录组学筛选miR-125b-2敲除小鼠睾丸发育相关基因及信号通路的研究

Screening of Genes and Signaling Pathway Related to Testicular Development in miR-125b-2 Knockout Mouse Based on Transcriptomics

旨在研究miR-125b-2对动物精子发生和成熟的调节作用,挖掘与miR-125b-2有密切靶向关系的功能基因及信号通路。本研究利用CRISPR/Cas9基因编辑技术,构建了miR-125b-2基因敲除小鼠模型。将3只野生型(WT)小鼠和3只miR-125b-2敲除型(KO)小鼠睾丸的总RNA样分别制成样品池,采用Illumina HiSeq TM 4000平台进行转录组测序(RNA-seq),将组装得到的Unigene序列注释到Nr和KEGG数据库中注释,并进行睾丸差异表达基因(DEGs)聚类分析。结果显示,在WT和KO组睾丸组织中共筛选出324个DEGs,其中被GO注释的180个DEGs显著富集到80个包括精子染色质缩合在内的生物学过程,22个含有线粒体内膜预序列转位酶复合物在内的细胞组成以及41个包括核糖体结构组成在内的分子功能。同时KEGG分析显示,所有被注释的DEGs显著富集到74条信号通路,其中排前3位极显著的信号通路依次为:RNA转运、信使RNA监视通路和合硒化合物新陈代谢。综上表明,敲除miR-125b-2主要影响睾丸中与精子染色质结构和线粒体功能相关基因的表达,其中与精子发生相关的3个基因Papolb、Tssk 1和Slc 22a14表达均显著上调。结果为进一步研究miR-125b-2对精子生成的功能调节提供基础。

The objective of this study was to investigate the regulation of miR-125b-2 on spermatogenesis and sperm maturation in animals and find the target genes of miR-125b-2 and related signaling pathways.In this study,the miR-125b-2 knockout mouse model was established by using CRISPR/Cas9.Three wild type(WT)and three miR-125b-2 knockout(KO)mice were selected and dissected,followed by testis tissues collection.Their total RNA were extracted and mixed into sample pool,respectively.The transcriptomes were analyzed by Illumina HiSeq TM 4000 RNA-Seq technology.All of Unigene sequences were annotated using the Blast search both in Nr and KEGG database.The differentially expressed genes(DEGs)in testis were conducted into cluster analysis.The results showed that 324 DEGs were identified,and 180 DEGs were annotated.GO analysis results showed that the 180 DEGs were significantly enriched in 80 biological process items including sperm chromatin condensation,22 cellular component items including mitochondrial inner membrane presequence translocase complex,and 41 molecular function terms including structural constituent of ribosome.KEGG analysis results indicated all annotated DEGs were significantly enriched in 74 signaling pathways,in which the top 3 pathways were RNA transport,mRNA surveillance pathway and selenocompound metabolism.In conclusion,all DEGs identified between WT and KO mice testes were mainly involved in sperm chromatin and mitochondria function.Especially,the expression levels of 3 spermatogenesis related genes,(Papolb,Tssk 1 and Slc 22a14)were significantly increased.The results provide a basis for further study on the function of miR-125b-2 in regulation of spermatogenesis process.