MicroRNA sequence analysis of plasma exosomes in early Legg-Calvé-Perthes disease

Abstract

The pathogenesis of Legg-Calvé-Perthes disease (LCPD) has not been fully elucidated, and studies on epigenetic changes that may contribute to the pathogenesis of LCPD are rare. MicroRNAs (miRNAs) are epigenetic modifications that play a critical role in gene regulation. This study aimed to determine the expression profiles of circulating exosomal miRNAs and examine the role of exosomal miRNAs in LCPD. Exosomes were extracted from the plasma of three patients with LCPD and three matched healthy volunteers. Total exosomal miRNAs were isolated, and next-generation sequencing and bioinformatic approaches were performed. The top 10 most differentially upregulated miRNAs were identified, and qRT-PCR validation was performed using additional 10 matches. In Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, plasma exosomes were used in verifying osteoclastogenesis and the endothelial dysfunction phenotypes involved. The elevated miRNAs in LCPD plasma exosomes were tested for osteoclastogenesis and endothelial dysfunction in vitro. Sequencing results revealed the expression profiles of plasma exosomal miRNAs with differential expression from the DESeq-identified miRNA profiles in LCPD versus controls in a pairwise comparison. Gene Ontology and KEGG pathway analyses indicated that the predicted target genes of different miRNAs were mainly enriched in the endothelial and osteoclast cells related to signaling pathways. Functional phenotype experiments showed that the plasma exosomes in the LCPD group promoted osteoclastogenesis and endothelial cell dysfunction. qRT-PCR experiments showed that nine miRNAs in circulating exosomes in LCPD patients were higher than those in the healthy controls. miR-3133, miR-4644, miR-4693-3p, and miR-4693-5p promoted endothelial dysfunction, and miR-3133, miR-4693-3p, miR-4693-5p, miR-141-3p and miR-30a promoted osteoclastogenesis in vitro. This study demonstrated that plasma exosomes from LCPD promote endothelial cell dysfunction and osteoclastogenesis likely through their miRNAs, which might contribute to the development of LCPD.

Keywords: Endothelial dysfunction; Exosome; Legg–Calvé–Perthes disease; Osteoclastogenesis; microRNA.