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Background The altered expression of circulating miRNAs has been discovered in many autoimmune diseases (ADs). With rare existing research, it is still unclear in Hashimoto's thyroiditis (HT). We detected plasma miRNA expression of HT patients in this three-stage designed study. Methods Differently expressed miRNAs (4 HT pools vs. 1 normal control pool) were identified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) based Exiqon panel (miRCURY-Ready-to-Use- PCR-Human- panel-I + II-V1.M) in the initial discovery stage. These miRNAs were then confirmed in the training stage and further validated in the testing stage using qRT-PCR with 64 (32 HT vs. 32 NCs) and 136 samples (68 HT vs. 68 NCs), respectively. Results A total of 10 miRNAs showed differential expression through the training stage. For further validation in the testing stage, expression of 6 miRNAs ( miR - 205 , miR - 20a - 3p , miR - 375 , miR - 296 , miR - 451 , miR - 500a ) were consistent with those in the training stage. Combination results showed that these 6 miRNAs were significantly up-regulated in peripheral plasma of HT patients compared with normal controls ( P < 0 . 05 ). In addition, the six-miRNA signature was evaluated to be a potential diagnostic marker of HT. The areas under the receiver operating characteristic curve of the signature were 0.80, 0.75 and 0.69 for the training, testing and the combined stages, respectively. Three miRNAs were associated with TSH levels in HT patients ( miR - 451 , P = 0.043; miR - 375 , P = 0.043; miR - 500a , P = 0.043). Additionally, miR - 20a - 3p was related with TgAb level ( P = 0.046). Conclusions We identified a miRNA signature including six dysregulated plasma miRNAs which could act as a diagnostic marker in plasma of HT, providing more evidence and better understanding for the association between circulating miRNAs and autoimmune diseases. [ABSTRACT FROM AUTHOR]