Differential Expression Profiles of Plasma Exosomal microRNAs in Rheumatoid Arthritis

Abstract

Aim: Differential expression maps of microRNAs (miRNAs) are connected to the autoimmune diseases. This study sought to elucidate the expression maps of exosomal miRNA in plasma of rheumatoid arthritis (RA) patients and their potential clinical significance.

Methods: In the screening phase, small RNA sequencing was performed to characterize dysregulated exosome-derived miRNAs in the plasma samples from six patients with RA and six healthy patients. At the independent verification stage, the candidate plasma exosomal miRNAs were verified in 40 patients with RA and 32 healthy patients by using qRT-PCR. The correlation of miRNA levels and clinical characteristics was tested in patients with RA. The value of these miRNAs in diagnosing RA was assessed with the receiver operating characteristic curve.

Results: During the screening phase, 177 and 129 miRNAs were increased and decreased in RA patients and healthy controls, respectively. There were 10 candidate plasma exosomal miRNAs selected for the next identification. Compared with the healthy controls, eight plasma exosomal miRNAs (let-7a-5p, let-7b-5p, let-7d-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-128-3p, and miR-25-3p) were significantly elevated in RA patients, but miR-144-3p and miR-15a-5p expression exhibited no significant changes. The let-7a-5p and miR-25-3p levels were linked to the rheumatoid factor-positive phenotype in RA patients. For the eight miRNAs, the area under the subject work characteristic curve (AUC) is 0.641 to 0.843, and their combination had a high diagnostic accuracy for RA (AUC = 0.916).

Conclusion: Our study illustrates that novel exosomal miRNAs in the plasma may represent potential noninvasive biomarkers for RA.

Keywords: biomarker; microRNAs; plasma exosome; rheumatoid arthritis; small RNA sequencing.

Figure 1

Identification of plasma exosomes. (a) TEM photograph of plasma exosomes; (b) NanoFCM of plasma exosomes; (c) results of exosome markers of plasma exosomes by WB.

Figure 2

Identification of differential expressed exosomal miRNAs. (a) Clustering hierarchy of the differential expressed exosomal miRNAs between RA patients and the healthy control subjects. Red rectangles indicate high levels of expression, and blue rectangles indicate low levels of expression; (b) volcano plot analysis of differential expression of exosomal miRNAs between RA patients and the healthy control subjects. The red plots indicate upregulated gene expression, the grey plots indicate nonsignificant genes, and the green plots represent genes that were downregulated.

Figure 3

KEGG pathway analysis of the differentially expressed exosomal miRNAs. (a) Upregulated; (b) downregulated.

Figure 4

Targets of upregulated and downregulated differentially expressed exosomal miRNAs according to the GO analysis. Enriched GO terms in the molecular function (a), cellular component (b), and biological process (c) categories.

Figure 5

(a–j) Comparison of relative levels of 10 plasma exosomal miRNAs between RA patients and control subjects.

Figure 6

(a–j) Comparison of relative levels of 10 plasma exosomal miRNAs between the stable and active RA patients.

Figure 7

ROC curves of eight plasma exosomal miRNAs and combined miRNAs for RA diagnosis.