Characteristics of circular RNA expression of pulmonary macrophages in mice with sepsis-induced acute lung injury

Abstract

Circular RNAs (circRNAs) make up a large class of non-coding RNAs and play important roles in the pathology of a variety of diseases. However, their roles in pulmonary macrophage polarization after sepsisinduced lung injury is unknown. In this study, mice were divided into two groups: Sham control group and cecal ligation and puncture (CLP)-induced ALI group. Macrophages were isolated from lung homogenates 24 hours after SCLP/CLP. We started with RNA-seq of circRNA changes in macrophages and validated by RT-PCR in the following experiments. A total of 4318 circRNAs were detected in the two groups. Of these, 11 and 126 circRNAs were found to be significantly upregulated and downregulated, respectively, compared to the control (p≤0.05, Fold Change ≥2). Differentially expressed circRNAs with a high foldchange (fold-change >4, P<0.05) were selected for validation by qRT-PCR, 10 of which were verified. Furthermore, the most differentially expressed circRNAs within all the comparisons were annotated in detail with circRNA/miRNA interaction information using miRNA target prediction software. The network of circRNA-miRNA-mRNA was illustrated by cytoscape software. Gene ontology analyses indicated the upregulated circRNAs were involved in the multiple biological functions such as regulation of mitochondrion distribution and Notch binding, while the down-regulated circRNAs mainly involved in the biological process as histone H3K27 methylation. KEGG pathway analysis revealed TGF-beta signaling pathway was related to the upregulated circRNAs. The present study provides a novel insight into the roles of circRNAs in pulmonary macrophage differentiation and polarization post septic lung injury.

Keywords: CircRNAs; acute lung injury; macrophage polarization; miRNA; sepsis.

Figure 1

CircRNA expression profile of pulmonary macrophages in Sham versus CLP mice. A, Lungs were fixed, sectioned and stained with H&E. Representative sections are shown from sham and CLP mice (Original magnification, × 200). B, Evaluation of wet/dry weight ratio is compared between sham and CLP mice. All data are expressed as mean ± SEM (N = 8, *P < .05 vs Sham group). C, Macrophages in the lung homogenates were isolated and phenotyped by FACS analysis. Representative dot plots are shown for expression of percentage INOS and CD80 and their expressions on the F4/80 + macrophages in the lung homogenates. D, The percentage of F4/80 + INOS +and F4/80 + CD80 +macrophages were compared between the sham and CLP mice. All data are expressed as mean ± SEM (N = 4, *P < .05 vs Sham group). E, Volcano plot showing the differentially expressed circRNAs in the two groups [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 < .05 between the two groups of macrophages. F, Hierarchical cluster analysis revealed the expression profile of the dysregulated circRNAs in the two groups. G, The scatter plot shows the circRNA expression variation between the two groups. The values of X and Y axes in the scatter plot are the averaged normalized signal values of groups of samples (log2 scaled). The green lines are fold change lines. The circRNAs above the top green line and below the bottom green line indicated more than 1.5‐fold change of circRNAs between the two groups of samples

Figure 2

Validation of circRNA expression by RT‐PCR and network of circular RNAs and the predicted binding miRNAs. A, Changes in CircRNA expression were confirmed using RT‐PCR for select circRNAs in the Sham and CLP groups. Bars represent mean ± SEM (n = 3), *P < .05. B, The three up‐regulated circRNAs were annotated in detail according to the circRNA/miRNA interaction information by Cytoscape. Based on the miRNA prediction and bioinformatics analyses, we show the top 5 miRNAs may be regulated by three up‐regulated circRNAs and top 5 target genes of each miRNA, respectively