MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

Background
Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients.
Methods
miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets.
Results
Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology.
Conclusion
This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.

【背景】慢性疲劳综合征（CFS/ME）是一种病因未明的复杂多系统疾病，全球约1%的人群会出现该疾病导致的衰弱性症状。尽管已有大量研究证实CFS/ME患者体内存在广泛的基因表达异常，但目前尚不明确调控基因翻译的微小RNA（miRNA）是否参与了疾病的发病机制。本研究假设，患者白细胞内的微小RNA表达变化参与了CFS/ME的病理过程，因此这类分子可作为潜在的诊断生物标志物，在CFS/ME患者的外周血中被检测到。
【方法】本研究采用安必昂生物芯片V1版（Ambion Bioarray V1），对CFS/ME患者与健康对照者的外周血单个核细胞（PBMC）中的微小RNA表达水平进行分析。将筛选得到的差异表达微小RNA通过实时定量逆转录PCR（qRT-PCR）进行验证，并在独立患者队列的分选血液白细胞亚群中开展重复验证实验。随后，将本研究筛选出的与CFS/ME相关的微小RNA转染至原代自然杀伤细胞（NK细胞）中，通过基因表达分析鉴定其靶基因。
【结果】芯片分析共筛选出34种差异表达的微小RNA，所有这些微小RNA均呈现上调表达。其中4种微小RNA的表达变化经qRT-PCR验证得到确认。对独立患者队列的PBMC样本按细胞类型进行分选后发现，NK细胞、B细胞与单核细胞中均存在微小RNA表达异常，其中以NK细胞的异常最为显著。将hsa-miR-99b与hsa-miR-330-3p转染至原代NK细胞后，观察到的基因表达变化符合NK细胞活化特征，但细胞毒性减弱，这提示NK细胞功能缺陷参与了CFS/ME的病理过程。
【结论】本研究证实CFS/ME患者外周血单个核细胞中存在微小RNA表达异常，这类分子有望成为潜在的诊断生物标志物。其中，NK细胞中微小RNA的表达失调程度最高，其靶基因与细胞活化及效应功能异常密切相关。