Microarray analysis of circular RNA expression patterns in polarized macrophages

Abstract

Circular RNAs (circRNAs) are generated from diverse genomic locations and are a new player in the regulation of post-transcriptional gene expression. Recent studies have revealed that circRNAs play a crucial role in fine-tuning the level of microRNA (miRNA)-mediated regulation of gene expression by sequestering miRNAs. The interaction of circRNAs with disease-associated miRNAs suggests that circRNAs are important in the pathology of disease. However, the effects and roles of circRNAs in macrophage polarization have yet to be explored. In the present study, we performed a circRNA microarray to compare the circRNA expression profiles of bone marrow-derived macrophages (BMDMs) under two distinct polarizing conditions (M1 macrophages induced by interferon-γ and LPS stimulation, and M2 macrophages induced by interleukin-4 stimulation). Our results showed that a total of 189 circRNAs were differentially expressed between M1 and M2 macrophages. Differentially expressed circRNAs with a high fold-change were selected for validation by RT-qPCR: circRNA-003780, circRNA-010056, and circRNA-010231 were upregulated and circRNA-003424, circRNA-013630, circRNA-001489 and circRNA-018127 were downregulated (fold-change >4, P<0.05) in M1 compared to M2, which was found to correlate with the microarray data. Furthermore, the most differentially expressed circRNAs within all the comparisons were annotated in detail with circRNA/miRNA interaction information using miRNA target prediction software. In conclusion, the present study provides novel insight into the role of circRNAs in macrophage differentiation and polarization.

Figure 1

Identification of ex vivo polarized M1 and M2 macrophages. Bone marrow-derived macrophages (BMDM) were isolated from BALB/c mice and treated with LPS (100 ng/ml) and interferon-γ (IFN-γ) (20 ng/ml) for M1 polarization or interleukin-4 (IL-4) (20 ng/ml) for M2 polarization. (A) F4/80 expression was evaluated by FACS analysis. (B) mRNA expression levels of M1 markers Nos2, TNF-α and IL-12; and M2 markers Arg-1, Ym-1 (Chi3l3), Fizz1 (Retnla) and Mrc-1 (CD206) were quantified by RT-q PCR. The data are expressed as the means ± SEM of three independent experiments.

Figure 2

Circular RNA (circRNA) microarray analysis of polarized macrophages. Bone marrow-derived macrophages (BMDMs) were isolated from BALB/c mice and cultured in the presence of LPS (100 ng/ml) plus interferon-γ (IFN-γ) (20 ng/ml) or interleukin-4 (IL-4) (20 ng/ml). circRNA microarray was performed to analyze differential circRNA expression in distinct polarized macrophages. (A) Volcano plots comparing the expression of circRNAs in M1 macrophages to M2 macrophages. [Plot of circRNA expression log₂-transformed fold-changes (x-axis) vs. -log₁₀ P-value (y-axis)]. The red dots represent the circRNAs having fold-changes >2.0 and P-values <0.05 between the two types of macrophages; P-value was calculated using the paired t-test. (B) Heat maps of circRNA expression fold-change discriminating M1 macrophages from M2 macrophages. Red indicates a higher fold-change and green indicates a smaller fold-change. The columns represent cirRNAs in the two groups of macrophages while the rows are the significant fold-change of the circRNAs. (C) Confirmation of the differential expression of circRNAs by RT-qPCR. Seven differentially expressed circRNAs were validated by RT-qPCR. The y-axis represents the log₂-transformed median fold-change in expression. Data are expressed as the means ± SEM of three independent experiments.

Figure 3

A snippet of the detailed annotation for circRNA-010231/miRNA interaction. The circRNA/miRNA interaction was predicted with Arraystar's home-made miRNA target prediction software based on TargetScan and miRanda, and the most differentially expressed circRNAs within all the comparisons were annotated in detail with the circRNA/miRNA interaction information. Binding sites of conserved miRNAs with good mirSVR scores are represented.