Genome-wide identification and functional prediction of circular RNAs in response to heat stress in Chinese Holstein cows

Heat stress (HS) leads to substantial economic loss of dairy industry each year. The negative effect of HS in dairy cows is becoming one of the more urgent issue due to accelerating side-effects of global warming. Various genes are involved in HS response but the information about the role of noncoding RNAs, especially circular RNAs (circRNAs) is largely unknown. In our study, we aimed to investigate the different expression profile of circRNAs between HS and Non-heat-stressed condition (NC) of Chinese Holstein cow’s mammary gland. CircRNAs were identified using RNA sequencing and bioinformatics analysis. In total, 37405 circRNAs were detected and 95 were differentially expressed (DE), including 15 downregulated and 80 upregulated circRNAs in HS group compared to NC. Eight circRNAs were randomly selected to verify the RNA sequencing result. Further, Sanger sequencing validated the backsplicing site of the eight circRNAs. Moreover, results obtained from the Quantitative real time PCR (qRT-PCR) showed consistent expression trend with that of RNA sequencing. GO annotation and KEGG analysis suggested that these DE circRNAs probably involved in the energy metabolic regulation. Furthermore, we constructed ceRNA network and the result indicated that these DE circRNAs could regulate lactation through IGF1 and PRL signaling pathway.

热应激（Heat Stress, HS）每年都会给乳业造成巨额经济损失。随着全球变暖的负面影响日益加剧，热应激对奶牛的不良影响已成为愈发紧迫的问题之一。目前已有诸多基因被证实参与热应激应答，但针对非编码RNA（尤其是环状RNA，circular RNAs, circRNAs）在该过程中的作用，相关认知仍十分有限。本研究旨在探究中国荷斯坦奶牛乳腺组织在热应激组与非热应激组（Non-heat-stressed condition, NC）中的环状RNA差异表达谱。研究通过RNA测序与生物信息学分析完成环状RNA的鉴定工作，总计检测到37405条环状RNA，其中95条为差异表达（differentially expressed, DE）环状RNA：与非热应激组相比，热应激组中有15条表达下调、80条表达上调。研究随机选取8条环状RNA对RNA测序结果进行验证，随后通过桑格测序（Sanger sequencing）验证了这8条环状RNA的反向剪接位点。此外，实时荧光定量PCR（Quantitative real time PCR, qRT-PCR）得到的表达趋势与RNA测序结果完全一致。基因本体（Gene Ontology, GO）注释与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析结果显示，这些差异表达环状RNA可能参与能量代谢调控。本研究进一步构建了内源竞争RNA（competing endogenous RNA, ceRNA）调控网络，结果表明这些差异表达环状RNA可通过胰岛素样生长因子1（IGF1）信号通路与催乳素（PRL）信号通路调控泌乳过程。