Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
Viruses. 2021 May 23;13(6):967. doi: 10.3390/v13060967.
MicroRNAs (miRNAs) are essential regulators of gene expression in humans and can control pathogenesis and host-virus interactions. Notably, the role of specific host miRNAs during influenza virus infections are still ill-defined. The central goal of this study was to identify novel miRNAs and their target genes in response to influenza virus infections in airway epithelium. Human airway epithelial cells exposed to influenza A virus (IAV) induced several novel miRNAs that were identified using next-generation sequencing (NGS) and their target genes by biochemical methods. NGS analysis predicted forty-two RNA sequences as possible miRNAs based on computational algorithms. The expression patterns of these putative miRNAs were further confirmed using RT-PCR in human bronchial epithelial cells exposed to H1N1, H9N1(1P10), and H9N1 (1WF10) strains of influenza virus. A time-course study showed significant downregulation of put-miR-34 in H1N1 and put-miR-35 in H9N1(1P10)-infected cells, which is consistent with the NGS data. Additionally, put-miR-34 and put-miR-35 showed a high fold enrichment in an argonaute-immunoprecipitation assay compared to the controls, indicating their ability to form a complex with argonaute protein and RNA-induced silencing complex (RISC), which is a typical mode of action found with miRNAs. Our earlier studies have shown that the replication and survival of influenza virus is modulated by certain transcription factors such as NF-ĸB. To identify the target(s) of these putative miRNAs, we screened 84 transcription factors that have a role in viral pathogenesis. Cells transfected with mimic of the put-miR-34 showed a significant decrease in the expression of Signal Transducers and Activators of Transcription 3 (STAT3), whereas the inhibitor of put-miR-34 showed a significant increase in STAT3 expression and its phosphorylation. In addition, put-miR-34 had 76% homology to the untranslated region of STAT3. NGS and PCR array data submitted to the Gene Ontology project also predicted the role of transcription factors modulated by put-miR-34. Our data suggest that put-miR-34 may be a good target for antiviral therapy.
微小 RNA（miRNAs）是人类基因表达的重要调控因子，能够控制发病机制和宿主-病毒相互作用。值得注意的是，特定宿主 miRNAs 在流感病毒感染中的作用仍未明确。本研究的主要目标是鉴定呼吸道上皮细胞感染流感病毒时的新型 miRNAs 及其靶基因。用下一代测序（NGS）和生化方法鉴定暴露于甲型流感病毒（IAV）的人呼吸道上皮细胞中诱导的几种新型 miRNAs。NGS 分析基于计算算法预测了 42 个可能的 miRNA 序列。用 RT-PCR 进一步证实了这些候选 miRNA 在人支气管上皮细胞中暴露于 H1N1、H9N1（1P10）和 H9N1（1WF10）流感病毒株时的表达模式。时间进程研究表明，H1N1 感染细胞中 put-miR-34 显著下调，H9N1（1P10）感染细胞中 put-miR-35 显著下调，这与 NGS 数据一致。此外，与对照相比，put-miR-34 和 put-miR-35 在 Argonaute 免疫沉淀试验中表现出高丰度富集，表明它们能够与 Argonaute 蛋白和 RNA 诱导沉默复合物（RISC）形成复合物，这是 miRNA 常见的作用模式。我们之前的研究表明，NF-ĸB 等某些转录因子调节流感病毒的复制和存活。为了鉴定这些候选 miRNA 的靶标，我们筛选了 84 种参与病毒发病机制的转录因子。转染 mimic 的细胞中 put-miR-34 的表达显著降低，而 put-miR-34 的抑制剂则显著增加 STAT3 的表达及其磷酸化。此外，put-miR-34 与 STAT3 的非翻译区有 76%的同源性。提交给基因本体项目的 NGS 和 PCR 阵列数据也预测了转录因子的作用受 put-miR-34 调节。我们的数据表明，put-miR-34 可能是抗病毒治疗的一个很好的靶点。
