Circular RNA Expression in the Lungs of Sickle Cell Disease Mice

Abstract

Rationale and Objective Circular RNA (circRNA), a novel class of endogenous non-coding RNA, can regulate gene expression through binding to miRNAs (sponge effect). Pulmonary hypertension (PH) is a serious complication of sickle cell disease (SCD) that causes significant morbidity and mortality. The role of circRNAs in SCD-PH pathogenesis has not been defined. We sought to explore the expression profiles of circRNAs and mRNAs in lungs of sickle cell (SS) mice that spontaneously develop PH. Methods and Results Total RNAs isolated from the lungs of 5 SS male mice and 5 littermate control (AA) mice (15-17 weeks old) were subjected to circRNA expression profiling using the Arrystar™ mouse circRNA array V2 and mRNA expression profiling using Arraystar™ mouse array. For circRNA analysis, Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software limma package. Volcano plot filtering was used to screen for differentially expressed (DE) circRNAs with statistical significance (Fold Change >1.5, p< 0.05). 21 circRNAs were upregulated (Up) and 127 circRNAs were downregulated (Dn) in lungs of SS mice compared to AA mice. For mRNA analysis, raw signal intensities were normalized in quantile method by GeneSpring GX software (Agilent Technologies). Volcano plot filtering was used to screen for DE mRNAs with statistical significance (Fold Change >2.0, p< 0.05). 1,753 mRNAs were upregulated and 1,455 mRNAs were downregulated in lungs of SS mice compared to AA mice. Database for Annotation, Visualization and Integrated Discovery bioinformatics analysis was used for Gene Ontology (GO) enrichment including biological process (BP), molecular function (MF), and cellular component (CC), and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database. GO analysis showed that the altered mRNAs were mainly distinct to negative (Up) or positive (Dn) regulation of biological processes in BP, ion binding (Up) or organic cyclic compound binding (Dn) in MF, and membrane-bounded organelle and vesicle (Up) and non-membrane-bounded organelle and intracellular organelle (Dn) in CC. Pathway analysis revealed that MAPK signaling pathway was significantly associated with upregulated mRNAs, whereas C-type lectin receptor signaling pathway was associated with downregulated mRNAs. circRNA-mRNA co-expression network analysis was evaluated by Coding-Non-coding gene Co-expression network analysis and generated by Cytoscape software (The Cytoscape Consortium). Conclusions These results identify new potential targets for therapeutic intervention in SCD and provide new insights into SCD pulmonary vascular dysfunction and PH pathogenesis.