High miR-451 expression in peripheral blood mononuclear cells from subjects at risk of developing rheumatoid arthritis

Abstract

Individuals carrying anti-citrullinated protein antibodies (ACPA) are considered at high risk of developing rheumatoid arthritis (RA). The altered expression of miRNAs contributes to the pathogenesis of RA. We aimed to identify differentially expressed miRNAs in the peripheral blood of ACPA-positive individuals with arthralgia at risk of RA compared to healthy controls (HC) and to determine their implications in the preclinical phase of RA. A comprehensive analysis of miRNAs revealed the dysregulation of miR-451 in peripheral blood mononuclear cells (PBMC) and plasma from RA-risk individuals. Higher miR-451 expression in PBMC from RA-risk individuals was further validated. Notably, miR-451 was previously shown to regulate CXCL16, a protein involved in RA pathogenesis. The expression of miR-451 in PBMC positively correlated with the CXCL16 mRNA, which could be secondary to the inflammation-induced expression of miR-451. Transfection of monocytes with pre-miR-451 in vitro resulted in the downregulation of CXCL16. Moreover, flow cytometry revealed a lower count of CXCL16-positive monocytes in RA-risk individuals. We propose that the constitutive or inflammation-induced upregulation of miR-451 in PBMC downregulates the expression of CXCL16, reduces the inflammatory milieu and thereby strives to delay the shift from the preclinical phase to the clinical manifestation of RA. This hypothesis warrants further investigation.

Figure 1

Workflow of our study. In Phase I, miRNAs in plasma and PBMC from ACPA-positive individuals with arthralgia at risk of developing RA and healthy controls (HC) were analysed. Phase II included a further analysis of target genes of selected miRNAs.

Figure 2

Expression of miR451 in (A) PBMC and (B) plasma from healthy controls (HC) and RA-risk individuals with arthralgia in the validation cohort using single assays. NS, not significant.

Figure 3

(A) Higher expression of the CXCL16 mRNA in PBMC from RA-risk individuals with arthralgia compared to HC. (B) A positive correlation between CXCL16 and miR-451 expression in PBMC from RA-risk individuals with arthralgia was observed. (C) CXCL16 expression was not correlated with miR-451 expression in PBMC from HC.

Figure 4

Analysis of CXCL16 levels in PBMC and plasma. (A) Plasma levels of CXCL16 were not different between HC and RA-risk individuals. (B) The negative correlation between plasma levels of CXCL16 and circulating miR-451 levels in RA-risk individuals with arthralgia. (C) Flow cytometry gating strategy and identification of the transmembrane form of CXCL16 in monocytes and NK cells. (D) Lower count of CXCL16-positive monocytes in RA-risk individuals with arthralgia compared to HC. (E) No differences in the numbers of CXCL16-positive NK cells were observed between RA-risk individuals with arthralgia and HC. NS, not significant. The gating strategy was created in Summit V4.3.01 software (DAKO Cytomation, Fort Collins, CO, USA).

Figure 5

Regulation of miR-451 and CXCL16 in monocytes. Transfection of monocytes with pre-miR-451 downregulates the expression of CXCL16 at the (A) mRNA and (B) protein levels. (C) Stimulation of monocytes with CXCL16 tends to upregulate miR-451.