Transcriptome microRNA profiling of bovine mammary epithelial cells challenged with Escherichia coli or Staphylococcus aureus bacteria reveals pathogen directed microRNA expression profiles. Bos taurus

Purpose: The goal of this study is to explore the role of miRNAs in dairy cow response to E. coli and S. aureus, mastitis causing pathogens, is not well understood. Results: The global expression of miRNAs in bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing. A total of 231 known bovine miRNAs were identified with more than 10 counts per million (CPM) in at least one of 13 libraries and 5 miRNAs including bta-miR-21-5p, miR-27b, miR-22-3p, miR-184 and let-7f represented more than 50% of the total reads of known bovine miRNAs. One hundred and fifty novel miRNAs were identified and half of them belong to the bta-miR-2284 family. Seventeen miRNAs were significantly (P<0.05) differentially regulated by the presence of pathogens. E. coli initiated an earlier regulation of miRNAs (6 miRNAs differentially regulated within the first 6 hrs post challenge as compared to one for S. aureus) while S. aureus presented a delayed response. Five differentially expressed miRNAs (Bta-miR184, miR-24-3p, miR-148, miR-486 and bta-let-7a-5p) were unique to E. coli while four (bta-miR-2339, miR-499, miR-23a and miR-99b) were unique to S. aureus. In addition, our study revealed a temporal differential regulation of five miRNAs (bta-miR-193a-3p, miR-423-5p, miR-30b-5p, miR-29c and miR-un116) in unchallenged cells. Target gene predictions of pathogen differentially expressed miRNAs indicate a significant enrichment in gene ontology functional categories in development/cellular processes, biological regulation as well as cell growth and death. Furthermore, target genes were significantly enriched in several KEGG (Kyoto encyclopedia of genes and genomes) pathways of the immune system, signal transduction, cellular process, nervous system, development and pathways in human diseases, especially cancer. Conclusion: Using next-generation sequencing, our study identified 150 novel bovine miRNAs and revealed a pathogen directed differential regulation of miRNAs in MAC-T cells with roles in immunity and development. E. coli elicited an earlier differential regulation of miRNAs as opposed to a delayed regulation by S. aureus. Furthermore, target gene prediction showed significant enrichments for functions in different biological and cellular processes as well as KEGG pathways in immunity, development and human diseases. Our study provides a further confirmation of the involvement of mammary epithelia cells in contributing to the immune response to infecting pathogens and suggests the potential of miRNAs to serve as biomarkers for diagnosis of mastitis and development of control measures. Overall design: Bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing, no replicates, using illumina HiScanSQ platform.

**研究目的**：乳腺炎致病菌大肠杆菌（Escherichia coli, E. coli）与金黄色葡萄球菌（Staphylococcus aureus, S. aureus）感染奶牛后的免疫应答过程中，微小核糖核酸（miRNAs）所发挥的调控作用尚未被充分阐明。本研究旨在探究该作用机制。

**研究结果**：本研究采用高通量测序技术，对经热灭活金黄色葡萄球菌（S. aureus）或大肠杆菌（E. coli）刺激的牛乳腺上皮细胞（MAC-T细胞）以及未受刺激的对照组细胞中的miRNAs全局表达谱进行分析。刺激组处理时间分别为6、12、24、48小时，对照组采样时间点为0、6、12、24、48小时。本研究共鉴定出231个已知牛源miRNAs，其在至少1个测序文库中的每百万reads计数（CPM）均大于10；其中bta-miR-21-5p、miR-27b、miR-22-3p、miR-184及let-7f这5个miRNAs的总reads数占已知牛源miRNAs总reads数的50%以上。同时鉴定出150个全新牛源miRNAs，其中半数隶属于bta-miR-2284家族。共有17个miRNAs在致病菌刺激下呈现显著差异表达（P<0.05）。大肠杆菌可更早引发miRNAs的差异表达：刺激后6小时内即有6个miRNAs出现表达差异，而金黄色葡萄球菌仅1个，呈现出滞后的应答模式。其中5个差异表达miRNAs（Bta-miR184、miR-24-3p、miR-148、miR-486及bta-let-7a-5p）仅在大肠杆菌刺激组中出现表达差异，另有4个miRNAs（bta-miR-2339、miR-499、miR-23a及miR-99b）仅特异性表达于金黄色葡萄球菌刺激组。此外，本研究还发现，在未受致病菌刺激的对照组细胞中，5个miRNAs（bta-miR-193a-3p、miR-423-5p、miR-30b-5p、miR-29c及miR-un116）呈现出随时间变化的差异表达模式。对致病菌刺激下差异表达miRNAs的靶基因进行预测分析发现，其靶基因显著富集于基因本体（GO）功能分类中的发育/细胞过程、生物调控以及细胞生长与死亡通路。此外，靶基因还显著富集于京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）的多个通路类别，包括免疫系统、信号转导、细胞过程、神经系统、发育通路以及人类疾病相关通路（尤以癌症通路最为显著）。

**研究结论**：本研究通过高通量测序技术，共鉴定出150个全新牛源miRNAs，并揭示了不同致病菌诱导MAC-T细胞中miRNAs呈现特异性差异表达的现象，该差异表达与免疫及发育调控密切相关。大肠杆菌可更早引发miRNAs的差异表达，而金黄色葡萄球菌则呈现滞后的调控模式。靶基因预测分析显示，这些差异miRNAs的靶基因显著富集于多种生物与细胞过程功能类别，以及免疫、发育和人类疾病相关的KEGG通路。本研究进一步证实了乳腺上皮细胞参与宿主对致病菌的免疫应答，并提示miRNAs有望作为奶牛乳腺炎诊断标志物及防控措施开发的潜在靶点。

**实验设计**：本研究采用Illumina HiScanSQ测序平台，通过高通量测序对经热灭活金黄色葡萄球菌（S. aureus）或大肠杆菌（E. coli）刺激的牛乳腺上皮细胞（MAC-T细胞）以及未受刺激的对照组细胞的miRNAs表达谱进行分析。刺激组处理时间分别为6、12、24、48小时，对照组采样时间点为0、6、12、24、48小时，所有样本均未设置生物学重复。