High-Throughput Sequencing Reveals Circulating miRNAs as Potential Biomarkers for Measuring Puberty Onset in Chicken (<i>Gallus gallus</i>)

There are still no highly sensitive and unique biomarkers for measurement of puberty onset. Circulating miRNAs have been shown to be promising biomarkers for diagnosis of various diseases. To identify circulating miRNAs that could be served as biomarkers for measuring chicken (Gallus gallus) puberty onset, the Solexa deep sequencing was performed to analyze the miRNA expression profiles in serum and plasma of hens from two different pubertal stages, before puberty onset (BO) and after puberty onset (AO). 197 conserved and 19 novel miRNAs (reads > 10) were identified as serum/plasma-expressed miRNAs in the chicken. The common miRNA amounts and their expression changes from BO to AO between serum and plasma were very similar, indicating the different treatments to generate serum and plasma had quite small influence on the miRNAs. 130 conserved serum-miRNAs were showed to be differentially expressed (reads > 10, P < 0.05) from BO to AO, with 68 up-regulated and 62 down-regulated. 4829 putative genes were predicted as the targets of the 40 most differentially expressed miRNAs (|log2(fold-change)|>1.0, P < 0.01). Functional analysis revealed several pathways that were associated with puberty onset. Further quantitative real-time PCR (RT-qPCR) test found that a seven-miRNA panel, including miR-29c, miR-375, miR-215, miR-217, miR-19b, miR-133a and let-7a, had great potentials to serve as novel biomarkers for measuring puberty onset in chicken. Due to highly conserved nature of miRNAs, the findings could provide cues for measurement of puberty onset in other animals as well as humans.目前仍缺乏用于检测青春期启动的高灵敏度、高特异性生物标志物。已有研究证实，循环微小RNA（circulating microRNA, miRNA）可作为多种疾病诊断的潜在生物标志物。为筛选可作为检测鸡（Gallus gallus）青春期启动生物标志物的循环miRNA，本研究采用Solexa高通量测序（Solexa deep sequencing）技术，分析了处于两个不同青春期阶段的母鸡血清与血浆中的miRNA表达谱：青春期启动前（BO）和青春期启动后（AO）。本研究共鉴定出197种保守型miRNA与19种新型miRNA（测序读段数>10），这些miRNA可在鸡的血清/血浆中表达。血清与血浆中共有的miRNA数量，以及其从青春期启动前到启动后的表达变化趋势均高度相似，表明制备血清与血浆的不同处理方式对miRNA的影响极小。在血清中，共有130种保守型miRNA在青春期启动前后呈现差异表达（测序读段数>10，P<0.05），其中68种表达上调，62种表达下调。针对40种差异表达最显著的miRNA（|log₂(倍数变化)|>1.0，P<0.01），本研究预测得到4829个潜在靶基因。功能分析显示，多条通路与青春期启动相关。进一步的实时荧光定量聚合酶链反应（quantitative real-time PCR, RT-qPCR）验证结果表明，由miR-29c、miR-375、miR-215、miR-217、miR-19b、miR-133a及let-7a组成的七miRNA组合，具备作为检测鸡青春期启动新型生物标志物的巨大潜力。鉴于miRNA的高度保守性，本研究结果可为其他动物乃至人类的青春期启动检测提供重要参考依据。