DataSheet_1_Small RNA Expression Profiling Reveals hsa-miR-181d-5p Downregulation Associated With TNF-α Overexpression in Sjögren’s Syndrome Patients.doc

MicroRNAs (miRNAs) are small non-coding RNAs (sRNA), that alter gene expression by binding to target messenger RNAs (mRNAs) and repressing translation. Dysregulated miRNA expression has been implicated in the pathogenesis of autoimmune diseases such as Sjögren’s syndrome (SS). The aim of this study was to characterize the global profile of sRNAs in labial salivary glands (LSG) from SS-patients and to validate potential miRNA candidates implicated in glandular inflammation. LSG from 21 SS-patients and 9 sicca controls were analyzed. A global next generation sequencing (NGS)-based sRNA profiling approach was employed to identify direct targets whereby differentially expressed miRNAs were predicted using bioinformatics tools. miRNA levels were validated by TaqMan and target mRNA levels were determined by quantitative real-time PCR. We also performed in vitro assays using recombinant TNF-α. NGS shows that ~30% of sRNAs were miRNAs. In comparison with samples from sicca controls, four miRNAs were found differentially expressed in LSG from SS-patients with low focus score (LFS) and 18 from SS-patients with high focus score (HFS). The miRNA with the most significant changes identified by NGS was hsa-miR-181d-5p and downregulation was confirmed by TaqMan analysis. Levels of TNF-α mRNA, a direct target of hsa-miR-181d-5p, were significantly increased and negatively correlated with hsa-miR-181d-5p presence. Moreover, positive correlations between TNF-α transcript levels, focus score, ESSDAI, and autoantibody levels were also detected. Furthermore, TNF-α stimulation decreased hsa-miR-181d-5p levels in vitro. Downregulation of hsa-miR-181d-5p in LSG from SS-patients could contribute to the glandular pro-inflammatory environment by deregulation of its direct target TNF-α. Further dissection of the pathophysiological mechanisms underlying the hsa-miR-181d-5p-mediated action in inflammatory conditions could be useful to evaluate the benefits of increasing hsa-miR-181d-5p levels for restoration of salivary gland epithelial cell architecture and function.

微小RNA（miRNAs）是一类小型非编码RNA（sRNA），可通过结合靶信使RNA（mRNA）并抑制翻译过程来调控基因表达。miRNA表达失调与干燥综合征（Sjögren’s综合征，SS）等自身免疫病的发病机制密切相关。本研究旨在明确干燥综合征患者唇腺（LSG）中小RNA的全局表达谱，并验证与腺体内炎症相关的潜在miRNA候选分子。本研究纳入21例SS患者与9例干燥对照者的唇腺样本进行分析。本研究采用基于下一代测序（NGS）的全局小RNA表达谱分析方法，通过生物信息学工具预测差异表达的miRNA并鉴定其直接靶标。通过TaqMan探针法验证miRNA的表达水平，通过实时荧光定量PCR检测靶mRNA的表达水平。本研究同时采用重组肿瘤坏死因子α（TNF-α）开展体外实验。NGS结果显示，约30%的小RNA为miRNA。与干燥对照者的样本相比，聚焦评分较低（LFS）的SS患者唇腺中发现4种差异表达的miRNA，而聚焦评分较高（HFS）的SS患者唇腺中则发现18种差异表达的miRNA。经NGS鉴定出变化最显著的miRNA为hsa-miR-181d-5p，其表达下调经TaqMan分析得到验证。作为hsa-miR-181d-5p的直接靶标，TNF-α mRNA的表达水平显著升高，且与hsa-miR-181d-5p的表达呈负相关。此外，本研究还发现TNF-α转录本水平、聚焦评分、欧洲干燥综合征疾病活动指数（ESSDAI）以及自身抗体水平之间存在正相关关系。进一步的体外实验显示，TNF-α刺激可降低hsa-miR-181d-5p的表达水平。SS患者唇腺中hsa-miR-181d-5p的表达下调，可通过影响其直接靶标TNF-α的表达，参与腺体内促炎微环境的形成。进一步阐明hsa-miR-181d-5p在炎症状态下的调控病理生理机制，有助于评估提高hsa-miR-181d-5p表达水平对修复唾液腺上皮细胞结构与功能的潜在价值。