High-throughput analysis of CircRNA in cows with naturally infected Staphylococcus aureus mammary gland

Circular RNAs (CircRNA) are a special type of non-coding RNA molecule with a closed ring structure and are not affected by RNA exonucases. It has stable expression, is not easy to degrade, and exists in most eukaryotes. However, circRNA regulation of cow mastitis has not been widely recognized. Mammary epithelial tissues were collected from healthy Holstein cows (HCN) and mastitis Holstein cows (HCU). RNA sequencing (RNA SEQ) was performed for the differentially expressed circRNAs, and analysis results showed that 19 differentially expressed circRNAs were identified in HCN and HCU, among which 6 circRNAs were up-regulated and 13 circRNAs were down-regulated. We randomly selected nine circRNAs for Q-PCR verification, and the results showed consistent expression. Three circRNAs: circRNA2860, circRNA5323 and circRNA4027 were confirmed to be significantly differentially expressed circRNAs in cow mastitis. Also, their host genes TRPS1, SLC12A2 and MYH11 might be directly or indirectly play a role in cow mastitis. Furthermore, RNA polymerase transcription factor binding and tight junction are most enriched in GO and KEGG pathways, respectively. In addition, the regulatory network of circRNA-miRNA has been inferred from a bioinformatics perspective, which may help to understand the underlying molecular mechanism of circRNAs involved in regulating mastitis in cows.

环状RNA（Circular RNAs，CircRNA）是一类特殊的非编码RNA分子，具有闭合环状结构，且不受RNA外切核酸酶（RNA exonucases）的降解影响。其表达稳定、不易降解，广泛存在于多数真核生物中。然而，环状RNA对奶牛乳腺炎的调控作用尚未得到广泛认可。本研究采集了健康荷斯坦奶牛（HCN）与患乳腺炎的荷斯坦奶牛（HCU）的乳腺上皮组织，对差异表达的环状RNA进行RNA测序（RNA-seq）。测序分析结果显示，在HCN与HCU样本中共鉴定出19个差异表达的环状RNA，其中6个表达上调，13个表达下调。本研究随机选取9个环状RNA进行实时定量聚合酶链反应（Q-PCR）验证，验证结果与测序表达趋势一致。其中circRNA2860、circRNA5323与circRNA4027这3个环状RNA被证实为奶牛乳腺炎相关的显著差异表达环状RNA，其宿主基因TRPS1、SLC12A2及MYH11可能直接或间接参与奶牛乳腺炎的发生发展过程。进一步分析显示，RNA聚合酶转录因子结合通路与紧密连接通路分别在基因本体（GO）与京都基因与基因组百科全书（KEGG）通路富集分析中呈现最高富集度。此外，本研究从生物信息学角度预测了circRNA-miRNA调控网络，这将有助于解析环状RNA参与调控奶牛乳腺炎的潜在分子机制。