以次世代定序及生物資訊分析經脂磷壁酸刺激人類嗜中性白血球之微小核醣核酸及影響標靶

Next-generation sequencing and bioinformatics analysis of miRNA and its targets in lipoteichoic acid treated Neutrophils

林佳蓉(Chia-Jung Lin)

指導教授 ： 陳昭文

高雄醫學大學/醫學院/臨床醫學研究所碩士班/碩士(2020年)

摘要

Toll-like receptors (TLRs)為模式辨識受體 (Pattern-recognition receptors, PRRs)其中的一員，其與免疫細胞的互動已有許多研究成果發表，但是與TLR訊號相關的microRNA及基因之間的交互作用還沒有被發掘。在此研究中，利用健康捐贈者的周邊血液分離出嗜中性白血球，並用金黃葡萄球菌的脂磷壁酸(lipoteichoic acid, LTA)處理過後並培養16小時，萃取RNA後利用次世代定序法定序定量microRNA及mRNA，再以生物資訊方法分析。經過分析後，在LTA治療組及對照組中一共有290個表現量差異的基因，經基因體資料庫分析比對，發現這些基因與細胞遷移及運動的正調控、細胞成分移動的正調控、對外部刺激的反應、防禦反應、發炎反應、細胞表現接受器訊號路徑相關。除此之外，有38個具表現量差異的miRNA預估與訊號傳遞及細胞溝通的調控相關。有四個miRNA(hsa‐miR‐34a‐5p, hsa‐miR‐34c‐5p, hsa‐miR‐708‐5p, 及 hsa‐miR‐1271‐5p)和五個基因(MET, CACNB3, TNS3, TTYH3,及HBEGF) 之間的交互作用與LTA引導的相關訊號網息息相關。本研究為LTA刺激後健康嗜中性白血球的miRNA及其目標基因的互動及表現，提供了新的生物資訊見解。

關鍵字

次世代定序 ； 生物資訊 ； 脂磷壁酸 ； 嗜中性白血球

並列摘要

During facing infection, our immune system would be activated. As the regulation of toll-like receptors (TLRs) signaling, one class of pattern‐recognition receptors, and immune cell has been investigated by various study, the interaction of TLR signaling-activated microRNAs and genes has not been well studies. In this study, neutrophils were isolated from a healthy donor’s peripheral blood, and then treated with Staphylococcus aureus lipoteichoic acid (LTA) and incubated for 16 hrs. The total RNAs were extracted and the expression analysis of miRNAs and mRNAs were done and via next-generation sequencing and bioinformatic approaches. 290 differentially expressed genes between LTA-treated and vehicle-treated neutrophils were identified, and gene ontology analysis revealed some biological processes and pathways were enriched, including inflammatory responses, defense response, positive regulation of cell migration, motility, and locomotion, and cell surface receptor signaling pathway. In addition, 38 differentially expressed miRNAs were identified and predicted to be involved in regulating signal transduction and cell communication. The interaction of 4 miRNAs (hsa‐miR‐34a‐5p, hsa‐miR‐34c‐5p, hsa‐miR‐708‐5p, and hsa‐miR‐1271‐5p) and 5 genes (MET, CACNB3, TNS3, TTYH3, and HBEGF) was proposed to participate in the LTA‐induced signaling network. The present findings may provide novel information for understanding the detailed expression profiles and potential networks between miRNAs and their target genes in LTA‐treated healthy neutrophils.

並列關鍵字

NGS ； bioinformatics ； LTA ； Neutrophil

參考文獻

李曜珊. (2017). 簡介次世代定序技術及美國的法規管理. 當代醫藥法規月刊, 83, 1-12.

Alves-Filho, J. C., et al. (2009). Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis. Proc Natl Acad Sci U S A, 106(10), 4018-4023

Areschoug, T., and Gordon, S. (2008). Pattern recognition receptors and their role in innate immunity: focus on microbial protein ligands. Contrib Microbiol, 15, 45-60

Armstrong, J. J., et al. (1958). 882. Isolation and structure of ribitol phosphate derivatives (teichoic acids) from bacterial cell walls. Journal of the Chemical Society (Resumed)(0), 4344-4354

Backes, C., et al. (2016). miEAA: microRNA enrichment analysis and annotation. Nucleic Acids Res, 44(W1), W110-116