miR-183-5p Is a Potential Molecular Marker of Systemic Lupus Erythematosus

Abstract

Objective: To investigate microRNA (miRNA) expression profiles in individuals with systemic lupus erythematosus (SLE) and identify the valuable miRNA biomarkers in diagnosing and monitoring SLE.

Methods: Next-generation sequencing (NGS) was performed to assess miRNA amounts in peripheral blood mononuclear cells (PBMCs) from four SLE cases and four healthy controls. Quantitative polymerase chain reaction (qPCR) was carried out for validating candidate miRNAs in 32 SLE cases and 32 healthy controls. In addition, receiver operating characteristic (ROC) curve analysis was completed to evaluate diagnostic performance. Finally, the associations of candidate miRNAs with various characteristics of SLE were analyzed.

Results: A total of 157 miRNAs were upregulated, and 110 miRNAs were downregulated in PBMCs from SLE cases in comparison to healthy controls, of which the increase of miR-183-5p and decrease of miR-374b-3p were validated by qPCR and both showed good diagnostic performance for SLE diagnosis. Besides, miR-183-5p expression levels displayed a positive association with SLE disease activity index (SLEDAI) and anti-dsDNA antibody amounts.

Conclusion: Our data indicated that miR-183-5p is a promising biomarker of SLE.

Figure 1

Correlation heat map analysis. SLE and controls presented distinct clusters based on the miRNA expression profiles of the eight specimens except for SLE_F1.

Figure 2

Volcano plot and heat map analyses. (a) Totally, 157 and 110 miRNAs were upregulated and downregulated, respectively, in SLE cases compared with controls. (b) The differentially expressed miRNAs well differentiated SLE cases from healthy controls.

Figure 3

GO and KEGG enrichment analyses of the differentially expressed miRNAs. (a) GO enrichment analysis showed that the differentially expressed miRNAs were correlated with various biological processes such as molecular function, protein binding, and nucleotide binding activity. (b) KEGG enrichment analysis revealed the involvement of the differentially expressed miRNAs in various pathways such as cancer, MAPK signaling, and Rap1 signaling.

Figure 4

Expression levels of candidate miRNAs in SLE cases and controls in the validation phase. The expression level of miRNA was determined using qPCR from each group. Our data found that miR-183-5p was upregulated (a) and miR-374b-3p was downregulated (b). However, no significant difference was exhibited with regard to miR-1-3p (c) and miR-19b-3p (d). ns: p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01.

Figure 5

Diagnostic values of miR-183-5p and miR-374b-3p. ROC curve analysis indicated AUCs for miR-183-5p and miR-374b-3p of 0.703 (: 0.574–0.833) and 0.681 (95% CI: 0.542–0.82), respectively. Combination of miR-183-5p and miR-374b-3p yielded an AUC of 0.832 (95% CI: 0.727–0.937).

Figure 6

miR-183-5p is associated with patient data in SLE. The expression level of miR-183-5p was higher in SLE cases with nephritis (a) or arthritis (b) in comparison with negative SLE counterparts, showing significant positive associations with SLEDAI score (c) or anti-dsDNA antibody levels (d). ^∗p < 0.05.

Figure 7

Bioinformatics analysis of the potential target genes of miR-183-5p. (a) The potential target genes of miR-183-5p were predicted in Targetscan7.1, miRDB, and miRTarBase platforms. (b) PPI network analysis showed the Foxo1 with high degrees of connectivity in potential target genes.

Figure 8

miR-183-5p directly targets Foxo1 3′UTR. (a) The binding site of miR-183-5p and the position 226-242 of Foxo1 3′UTR wild-type (WT) and mutant-type (mut). (b) miR-183-5p ectopic expression significantly inhibited luciferase activity of the wild-type Foxo1 3′UTR reporter plasmid in comparison with the mutated counterpart. Groups labeled with different letters are statistically different from each other. ^∗p < 0.05. Differences between groups were analyzed for statistical significance by ANOVA with Fischer's probable least-square difference post hoc test.

Figure 9

Expression of Foxo1 in subjects and with miR-183-5p levels (a) Foxo1 amounts are reduced in SLE cases (n = 32) in comparison with healthy controls (n = 32). (b) Spearman's rank correlation revealed a negative relationship between Foxo1 amounts and miR-183-5p expression levels. ^∗p < 0.05; ^∗∗∗∗p < 0.0001.