Small RNA-seq analysis of circulating miRNAs in Cushing's syndrome

Patients with Cushing’s syndrome (Cushing's syndrome) in remission show sustained fatigue, myopathy, and an increased prevalence of sarcopenia. The mechanisms that determine these persistent muscle problems are not well known. We aimed to identify circulating microRNAs (miRNAs) with differential expression that could be potential biomarkers for the diagnosis and/or prognosis in Cushing's syndrome. In this data set, we analized thirty-six women in sustained remission for 13 ± 7 years (mean ± SD) from Cushing's syndrome, with a median age (IQ range) of 51 (45.2–60) years and mean ± SD BMI of 27 ± 4 Kg/m2, and 36 matched healthy controls using small RNA-sequencing of small RNA purified from plasma samples of these patients and control. In 7 patients sarcopenia was present according to the European Working Group on Sarcopenia in Older People (EWGSOP) criteria. Validation was carried out using RT-qPCR. For the validation, Taqman probes were performed on QuantStudio 5 equipment (Applied Biosystems). In a first discovery group using RNA-sequencing, plasma samples of 18 Cushing's syndrome patients and 18 healthy subjects were investigated; circulating miR-28-5p, miR-495-3p and miR-654-5p were upregulated in Cushing's syndrome patients as compared with controls (p<0.05). In a validation study of the 3 upregulated miRNAs in 36 patients and 26 controls, no differences were observed by RT-qPCR; however, the expression of circulating miR-28-5p was upregulated in Cushing's syndrome patients with sarcopenia as compared with those without (AUC for fold-change in the ROC analysis, 0.798; p=0.0156). The optimized cut-off value for miR-28-5p to identify Cushing's syndrome patients with sarcopenia was 3.80, which yielded a sensitivity of 86% and a specificity of 69%. Plama miRNA profiles of 18 Cushing's syndrome and 18 healthy subjects were generated by deep sequencing using Illumina NextSeq 550.