A comprehensive analysis of the circRNA–miRNA–mRNA network in osteocyte-like cell associated with Mycobacterium leprae infection

Bone formation and loss are the characteristic clinical manifestations of leprosy, but the mechanisms underlying the bone remodeling with Mycobacterium leprae (M. leprae) infection are unclear.

Methodology/Principal findings
Osteocytes may have a role through regulating the differentiation of osteogenic lineages. To investigate osteocyte-related mechanisms in leprosy, we treated osteocyte-like cell with N-glycosylated muramyl dipeptide (N.g MDP). RNA-seq analysis showed 724 differentially expressed messenger RNAs (mRNAs) and 724 differentially expressed circular RNA (circRNAs). Of these, we filtered through eight osteogenic-related differentially expressed genes, according to the characteristic of competing endogenous RNA, PubMed databases, and bioinformatic analysis, including TargetScan, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Based on these results, we built a circRNA–microRNA (miRNA)–mRNA triple network. Quantitative reverse-transcription polymerase chain reaction and western blots analyses confirmed decreased Clock expression in osteocyte-like cell, while increased in bone mesenchymal stem cells (BMSCs), implicating a crucial factor in osteogenic differentiation. Immunohistochemistry showed obviously increased expression of CLOCK protein in BMSCs and osteoblasts in N.g MDP–treated mice, but decreased expression in osteocytes.

Conclusions/Significance
This analytical method provided a basis for the relationship between N.g MDP and remodeling in osteocytes, and the circRNA–miRNA–mRNA triple network may offer a new target for leprosy therapeutics.

Author summary
Although the number of leprosy patients is gradually decreasing after the use of rifampin, those who have been cured still have bone damage. The Mycobacterium leprae is difficult to culture in vivo, and the potential biosafe problem should be considered. Thus, the minimal essential structure in Mycobacterium, N-glycosylated muramyl dipeptide, has been used to reveal the mechanism in leprosy. According to the analysis results, various differentially expressed genes have been filtered, and circadian Clock gene might be the key factor. We verified that Clock gene was upregulated in the process of osteogenesis. Osteocytes would maintain the balance between osteoblasts and osteoclasts in bone homeostasis. We noticed that the expression of Clock gene was decreased after treated with N-glycosylated muramyl dipeptide. This might be the reason why bone damage is still maintained in the cured patient, and regulating the expression of target at posttranscriptional level by microRNAs and circular RNAs can be the promising therapeutic method in leprosy bone damage.