乏情和发情初产母猪下丘脑-垂体轴中miRNA表达谱比较分析

Comparison and Analysis of miRNA Expression Profile in the Hypothalamic-pituitary Axis of Anestrous and Estrous Primiparous Sows

【目的】从神经内分泌系统揭示微小RNA(microRNA,miRNA)调控初产母猪情期活动的遗传机制。【方法】利用RNA-Seq技术对乏情和发情初产母猪下丘脑-垂体轴中的small RNA进行测序,获得了母猪下丘脑和垂体miRNA表达谱,并对筛选的差异表达miRNA进行GO和KEGG等功能分析。【结果】从所构建的4个文库中共鉴定出1096个miRNAs,其中已知miRNAs 364个,新预测miRNAs 732个。以发情母猪为对照,在乏情母猪的下丘脑有16个miRNAs存在差异表达,其中6个上调,10个下调;在乏情母猪垂体有9个miRNAs存在差异表达,其中2个上调,7个下调。下丘脑中16个差异表达miRNAs共预测到5212个靶基因,垂体中9个差异表达miRNAs共预测到6897个靶基因。下丘脑中靶基因主要在细胞投射组装、质膜结合的细胞投射组装、神经元投射发育的负调控、蛋白丝氨酸/苏氨酸激酶活性等GO条目及Ras信号通路、神经营养因子信号通路、ErbB信号通路、mTOR信号通路等KEGG信号通路中显著富集;垂体中靶基因主要在mRNA加工调控、Ras GTP酶结合、泛素样蛋白连接结酶活性、表皮生长因子受体结合等GO条目及蛋白激酶、鞘脂类信号通路、凋亡、ErbB信号通路、NF-κB信号通路等KEGG信号通路中显著富集。从构建的差异表达miRNAs与生殖相关功能靶基因的调控网络中,筛选的Chr3_8404_mature、ssc-miR-9和ssc-miR-34c可能在下丘脑-垂体轴中对初产母猪情期活动的调控起重要作用。下丘脑和垂体各选4个差异表达miRNAs进行实时荧光定量PCR验证,其表达趋势与测序结果基本一致。【结论】本研究成功构建了乏情和发情初产母猪下丘脑-垂体轴miRNA的表达谱,并对差异表达miRNA的功能进行了深度挖掘,筛选到了3个对初产母猪情期活动起重要作用的miRNAs,为初产母猪情期活动遗传机制的解析提供了miRNAs方面的理论依据。

【Objective】The purpose of this study was to reveal the genetic mechanism of microRNA(miRNA)on estrous activity in primiparous sows from the neuroendocrine system.【Method】RNA-Seq technology was utilized to sequence small RNA from the hypothalamic-pituitary axis of anestrous and estrous primiparous sows in this study.The miRNA expression profiles in the hypothalamus and pituitary of sows were obtained and the differentially expressed miRNA were screened for functional analysis such as GO and KEGG.【Result】A total of 1096 miRNAs were identified from four libraries,of which 364 were known miRNAs and 732 were novel miRNAs.Compared with estrous primiparous sows,16 differentially expressed miRNAs were identified in the hypothalamus of anestrous sows,including 6 up-regulated and 10 down-regulated miRNAs.9 differentially expressed miRNAs were identified in the pituitary of anestrous sows,including 2 up-regulated and 7 down-regulated miRNAs.A total of 5212 target genes were predicted for 16 differentially expressed miRNAs in the hypothalamus,and 6897 target genes were predicted for 9 differentially expressed miRNAs in the pituitary gland.Functional analysis of differentially expressed miRNA target genes in hypothalamus showed some GO entries and KEGG signaling pathways were significantly enriched,such as cell projection assembly,plasma membrane bounded cell projection assembly,negative regulation of neuron projection development,protein serine/threonine kinase activity,Ras signaling pathway,neurotrophin signaling pathway,ErbB signaling pathway,and mTOR signaling pathway,etc.These miRNA target genes in pituitary were also significantly enriched in some GO entries and KEGG signaling pathways,such as regulation of mRNA processing,Ras GTPase binding,ubiquitin-like protein ligase activity,epidermal growth factor receptor binding,protein kinase,sphingolipids signaling pathway,apoptosis,ErbB signaling pathway,and NF-κB signaling pathway,etc.Chr3_8404_mature,ssc-miR-9 and ssc-miR-34c were screened from miRNA regulatory network with target genes that were involved in regulation of reproduction,and these 3 miRNAs might play an important role in regulating estrous activity in the hypothalamic-pituitary axis of primiparous sows.8 differentially expressed miRNAs(4 in hypothalamus and 4 in pituitary)were selected for validation by Real-time quantitative PCR,and their expression trends were generally consistent with the sequencing results.【Conclusion】The miRNA expression profiles of hypothalamic-pituitary axis of anestrous and estrous primiparous sows were successfully constructed and the functions of differentially expressed miRNA were deeply explored in this study.3 miRNAs that play an important role in the estrous activity of primiparous sows were screened.A theoretical basis for the analysis of the genetic mechanism of estrous activity in primiparous sows had been provided.