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. 2017 Nov 10;19(1):249.
doi: 10.1186/s13075-017-1459-x.

Whole blood microRNA expression pattern differentiates patients with rheumatoid arthritis, their seropositive first-degree relatives, and healthy unrelated control subjects

Vidyanand Anaparti  1   2   3 , Irene Smolik  1   3   4 , Xiaobo Meng  1   2   3 , Victor Spicer  2 , Neeloffer Mookherjee  1   2   5 , Hani El-Gabalawy  6   7   8   9   10
Affiliations

Whole blood microRNA expression pattern differentiates patients with rheumatoid arthritis, their seropositive first-degree relatives, and healthy unrelated control subjects

Vidyanand Anaparti et al. Arthritis Res Ther. .

Abstract

Background: Epigenetic mechanisms can integrate gene-environment interactions that mediate disease transition from preclinical to clinically overt rheumatoid arthritis (RA). To better understand their role, we evaluated microRNA (miRNA, miR) expression profile in indigenous North American patients with RA who were positive for anticitrullinated protein antibodies; their autoantibody-positive, asymptomatic first-degree relatives (FDRs); and disease-free healthy control subjects (HCs).

Methods: Total RNA was isolated from whole blood samples obtained from HC (n = 12), patients with RA (n = 18), and FDRs (n = 12). Expression of 35 selected relevant miRNAs, as well as associated downstream messenger RNA (mRNA) targets of miR-103a-3p, was determined by qRT-PCR.

Results: Whole blood expression profiling identified significantly differential miRNA expression in patients with RA (13 miRNAs) and FDRs (10 miRNAs) compared with HCs. Among these, expression of miR-103a-3p, miR-155, miR-146a-5p, and miR-26b-3p was significantly upregulated, whereas miR-346 was significantly downregulated, in both study groups. Expression of miR-103a-3p was consistently elevated in FDRs at two time points 1 year apart. We also confirmed increased miR-103a-3p expression in peripheral blood mononuclear cells from patients with RA compared with HCs. Predicted target analyses of differentially expressed miRNAs in patients with RA and FDRs showed overlapping biological networks. Consistent with these curated networks, mRNA expression of DICER1, AGO1, CREB1, DAPK1, and TP53 was downregulated significantly with miR-103a-3p expression in FDRs.

Conclusions: We highlight systematically altered circulating miRNA expression in at-risk FDRs prior to RA onset, a profile they shared with patients with RA. Prominently consistent miR-103a-3p expression indicates its utility as a prognostic biomarker for preclinical RA while highlighting biological pathways important for transition to clinically detectable disease.

Keywords: Epigenetics; MicroRNA; Rheumatoid arthritis; Whole blood; miR-103a; miR-103a-3p; miRNA.

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Conflict of interest statement

Ethics approval and consent to participate

The biomedical research ethics board of the University of Manitoba approved the overall design of the study and consent forms (ethics, 2005:093; protocol, HS14453). Specific research agreements with the study communities were developed and approved by the community leadership. The conduct of the study was guided by the principles of community-based participatory research, a cornerstone of the Canadian Institutes of Health Research guidelines for aboriginal health research (http://www.cihr-irsc.gc.ca/e/29134.html). As such, community leadership provided input into the initial development of the project as well as ongoing input through advisory board meetings. Local healthcare providers were trained in study methodology and standard operating procedures. Regular knowledge translation activities such as newsletters and local radio appearances by study investigators provided the communities with updates regarding progress and significance. The study participants provided informed consent after the study was explained to them in detail, with the help of an INA translator from their community where necessary.

Consent for publication

Written consent was obtained from all the participants and authors of this study.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
a Unsupervised hierarchical clustering generated using fold change expression values of microRNAs (miRNAs, miRs) analyzed in patients with rheumatoid arthritis (RA) and first-degree relatives (FDRs) compared with healthy control subjects. Color scheme: Violet = increased expression; cyan = decreased expression; and white = unchanged. b Volcano plot showing expression of 33 miRNAs in the whole blood of patients with RA (closed squares) and FDRs (open triangles). The miRNAs that are significantly altered in both groups are located above the horizontal dashed line corresponding to P ≤ 0.05
Fig. 2
Fig. 2
a Kolmogorov-Smirnov (K-S) plots showing cumulative distribution of microRNA (miR) miR-103a-3p expression for healthy control subjects (HCs; open circles), patients with rheumatoid arthritis (RA; closed squares), and first-degree relatives (FDRs; open triangles). The distance between two distribution plots is represented by double arrows. K-S distance ≥ 0.5 was considered significant. b ROC curves (representing 1-specificity vs sensitivity values) for HCs, patients with RA, and FDRs, calculated using expression values of miR-103a-3p
Fig. 3
Fig. 3
Expression of microRNA (miR) miR-103a-3p in whole blood or peripheral blood mononuclear cells obtained from healthy control subjects (HCs), patients with rheumatoid arthritis (RA), or first-degree relatives (FDRs). miR-103a-3p transcriptional abundance was analyzed by qPCR. a Scatterplot of normalized cycle threshold (ΔC t) values and fold change of miR-103a-3p relative expression in whole blood of HCs (open circles), patients with RA (filled squares), and FDRs (open triangles). Error bars represent median values analyzed by Kruskal-Wallis test with Dunn’s test for multiple comparisons (*** P < 0.001 vs HCs). b Scatterplot of normalized ΔC t values showing miR-103a-3p expression in whole blood of HCs (open circles) and FDRs (open triangles) collected at two independent time points ~ 1 year apart (T1 and T2)
Fig. 4
Fig. 4
Transcript abundance of microRNA (miR) miR-103a-3p target messenger RNAs (DICER1, AGO1, CREB1, TP53, and DAPK1) analyzed by qPCR using total RNA obtained from whole blood of healthy control subjects (HCs), patients with rheumatoid arthritis (RA) and first-degree relatives (FDRs). Scatterplots represent relative fold change expression values compared with HCs. Error bars represent median values analyzed by Mann-Whitney U test. ***P < 0.001, ** P < 0.01, *P < 0.05. ns Nonsignificant compared with HCs
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