Table_1_MicroRNA profiling of cerebrospinal fluid from dogs with steroid responsive meningitis-arteritis and meningoencephalitis of unknown origin.xlsx

IntroductionNon-infectious inflammatory diseases of the central nervous system in dogs, such as steroid responsive meningitis-arteritis (SRMA) and meningoencephalitis of unknown origin (MUO), represent a common clinical challenge that needs extensive and multimodal work-up to reach a presumptive diagnosis. Both diseases are presumably caused by dysregulations of the immune system, but further research is needed in order to understand the molecular mechanisms behind each disease and to optimize treatment.
MethodsBy next-generation sequencing and subsequent quantitative real-time PCR (qPCR) verification, we designed a prospective case–control pilot study to analyze the small RNA profiles of cerebrospinal fluid from dogs suffering from MUO (N = 5), dogs suffering from SRMA (N = 8), and healthy dogs (N = 5) presented for elective euthanasia used as the Control group.
ResultsOur results showed an overall enrichment in Y-RNA fragments across all samples, followed by microRNAs (miRNAs) and ribosomal RNAs as the major findings. Additional traces of short RNA reads mapped to long non-coding RNAs and protein-coding genes were also found. From the detected canine miRNAs, miR-21, miR-486, miR-148a, miR-99a, miR-191 and miR-92a were among the most abundant. Dogs with SRMA showed higher differences in miRNA abundance than dogs with MUO when compared to healthy dogs, and miR-142-3p was consistently detected as differentially upregulated in both diseases, although at a low concentration. Moreover, miR-405-5p and miR-503-5p showed different profiles between SRMA and MUO dogs. Subsequent qPCR analyses confirmed miR-142-5p, miR-191-5p and miR-92a-3p as significantly upregulated miRNAs in dogs with SRMA and/or MUO.
DiscussionCerebrospinal fluid is a challenging biological material to use for profiling miRNAs due to the low content of circulating RNAs. Despite this, we could confirm several miRNAs being differentially abundant when comparing healthy dogs and dogs with MUO and SRMA, respectively. The results of this study indicate a potential role of miRNAs in the underlying molecular mechanisms of these diseases and establish the basis for further studies.

引言：犬类中枢神经系统非感染性炎症性疾病，如糖皮质激素反应性脑膜炎-动脉炎（steroid responsive meningitis-arteritis, SRMA）与未知病因性脑膜脑炎（meningoencephalitis of unknown origin, MUO），是临床常见诊疗难题，需通过多维度、全面的检查流程方可作出初步诊断。目前认为这两类疾病均由免疫系统失调引发，但仍需开展进一步研究以阐明其具体分子机制，并优化治疗方案。
方法：本研究通过二代测序（next-generation sequencing）结合后续实时定量聚合酶链反应（quantitative real-time PCR, qPCR）验证，设计了一项前瞻性病例对照预实验，对三类犬只的脑脊液小RNA表达谱进行分析：未知病因性脑膜脑炎患犬（N=5）、糖皮质激素反应性脑膜炎-动脉炎患犬（N=8），以及以择期安乐死的健康犬只作为对照组（N=5）。
结果：本研究所有样本均整体富集Y-RNA片段，其次为微小RNA（microRNAs, miRNAs）与核糖体RNA，此为主要发现。此外还检测到少量短RNA序列比对至长链非编码RNA及蛋白编码基因。在检测到的犬类微小RNA中，miR-21、miR-486、miR-148a、miR-99a、miR-191及miR-92a为丰度最高的分子之一。与健康犬相比，糖皮质激素反应性脑膜炎-动脉炎患犬的微小RNA表达丰度差异较未知病因性脑膜脑炎患犬更为显著；且miR-142-3p在两类疾病中均持续呈现上调差异表达，尽管其表达浓度较低。此外，miR-405-5p与miR-503-5p在糖皮质激素反应性脑膜炎-动脉炎患犬与未知病因性脑膜脑炎患犬中呈现不同的表达谱。后续qPCR分析证实，miR-142-5p、miR-191-5p及miR-92a-3p在糖皮质激素反应性脑膜炎-动脉炎和/或未知病因性脑膜脑炎患犬中显著上调。
讨论：由于循环RNA含量极低，脑脊液作为用于微小RNA表达谱分析的生物样本颇具挑战性。尽管如此，本研究仍证实，分别将健康犬与未知病因性脑膜脑炎患犬、糖皮质激素反应性脑膜炎-动脉炎患犬进行对比时，多种微小RNA的表达丰度存在显著差异。本研究结果提示微小RNA在这两类疾病的潜在分子机制中可能发挥作用，并为后续研究奠定了基础。