鸡性成熟启动特异性血清循环miRNA标记鉴定及其调控网络分析

Chicken Puberty was a complex biological process, its stages can not be assessed accurately and its molecular regulating mechanism remained ambiguous. Previous researches confirmed that miRNAs were accurate regulators of different developing events, and the serum circulating miRNAs were accurate inspectors of body state. Accordingly, this study used the Wenchang chicken, a meat-type breed with distinct precocious characters, as the material. Solexa sequencing method was used to detect the serum miRNA profiles in different stages near the puberty onset period. The different expressed miRNAs and their origins were identified by comparing to several databases. Then the special puberty onset circulating miRNA markers were identified through expression analysis by qRT-PCR methods. Colligating several biological information tools to predict the target genes of aimed miRNA markers. However, the special target genes were identified ultimately by analysis of mRNA common expression and luciferase report gene system. The controlling gene networks were also analyzed. The results could achieve accurate assessment of chicken puberty onset and processing stages, supply theories to interpret more perfect chicken puberty controlling mechanisms, contribute to selection and utilization of chicken puberty traits, and supply cues for puberty evaluation of other species as well as diagnosis of human precocious diseases.

鸡性成熟是一个复杂的生物学过程，启动时机及进程难以精确衡量，其分子调控机制目前尚不明确。研究证实miRNA不仅是动物发育事件的精细调节者，血清循环miRNA还是机体生物学和生理学状态的精细监测者。鉴于此，本研究拟以早熟性明显的文昌鸡为素材，采用solexa高通量测序技术检测性成熟启动前后不同时期血清循环miRNA表达谱，通过与不同数据库比对，探明差异表达miRNA及其起源；结合时序定量表达分析，鉴定出性成熟启动特异性血清循环miRNA标记。综合采用生物信息学工具预测标记miRNA靶基因，并通过标记miRNA与候选靶基因共表达分析、荧光素酶报告基因分析鉴定出特定靶基因及调控网络。研究结果将可实现鸡性成熟启动及进程的精确衡量，为从新的层面阐述更完善的鸡性成熟启动调控机制提供理论依据，提升鸡早熟性状的选育和利用水平，同时为其他物种早熟性评价及人类性早熟疾病临床诊断提供借鉴。

本研究选取早熟地方鸡品种文昌鸡为实验素材，开展了以下几个方面的研究：（1）测定不同发育时期母鸡性腺组织的发育性变化和血清LH浓度变化，度量性成熟启动时机。（2）利用Solexa高通量测序技术检测了性成熟启动前、后血清（浆）中miRNA表达谱，鉴定差异表达miRNA，并利用qPCR筛选性成熟启动度量特异性循环miRNA标记。同时，检测了性成熟启动前、后下丘脑中miRNA表达谱，探讨miRNA在鸡性成熟启动调控中的作用。（3）利用qPCR检测了部分miRNA靶基因在性成熟启动前、后下丘脑中的表达规律。.主要得出以下结论：（1）确定13周龄为文昌鸡母鸡的性成熟启动关键转折点，这为构建性成熟启动前、后高通量测序文库提供了前提。（2）通过高通量测序，在血清、血浆中鉴定出共表达miRNA 192个，血清特异性miRNA 1个，血浆特异性miRNA 4个，新miRNA 19个。相同发育时期血清与血浆中的miRNA表达丰度基本一致。鉴定出性成熟启动前、后血清中差异表达miRNA（P<0.05）有130个，其中上调68个，下调62个。qPCR检测表明9个候选miRNA在性成熟启动前期血清中表达水平基本一致，性成熟启动后miR-217-5p和miR-155的表达水平并未发生显著性变化，miR-29c-3p、miR-375、miR-215-5p、miR-9-5p、miR-19b-3p、miR-133a-3p和let-7a的表达水平显著升高，这7个候选miRNAs标记可用于文昌鸡性成熟启动时机的精确度量。鉴于miRNA功能的保守性，这些循环miRNA标记可以为其它动物性成熟启动度量及人类性早熟诊断提供借鉴。（3）通过高通量测序，在鸡下丘脑中鉴定出374个已知miRNA，144个已知miRNA的表达水平在性成熟启动前后发生了显著改变（P < 0.05）。15个差异最显著miRNA（P < 0.01）预测到2013个靶基因，qPCR检测表明其中7个时钟基因Per2、Bmal1/2、Clock、Cry1/2和Star的表达水平在性成熟启动后下丘脑中显著升高（P <0.01）。表明miRNA是参与鸡性成熟启动新的调节因子。（4）qPCR检测进一步证实两个靶基因调控通路c-Myc（↓）→LIN28（↓）┤let-7（↑）和POK（↑）┤PcG（↓）┤TRG（↑）与鸡性成熟启动调节相关。