Abstract: BACKGROUND: Multiple myeloma bone disease is a serious complication of multiple myeloma, and its pathogenesis is closely related to the dysregulation of the bone marrow microenvironment. Peripheral blood exosomes are secreted by various cell types and can reflect a variety of information such as the tumor microenvironment. Exosomal circular RNA is gradually becoming the focus of liquid biopsy due to its high stability, high abundance, high specificity, and high conservation. In multiple myeloma bone disease, there is still a lack of relevant research on the role of exosomal circular RNAs. With the development of single-cell RNA sequencing technology, clarifying the contribution of each cell type in the bone marrow microenvironment to multiple myeloma bone disease-related exosomal circular RNAs and the interaction between each cell type will help provide new biomarkers for the diagnosis and treatment of multiple myeloma bone disease.
OBJECTIVE: To preliminarily determine which cells in the bone marrow microenvironment the multiple myeloma bone disease-related peripheral blood exosomal circular RNAs originated from, and explore the effects of cell interactions in the bone marrow microenvironment on multiple myeloma bone disease at the single-cell level.
METHODS: Peripheral blood exosomes from six multiple myeloma bone disease patients and five healthy controls were collected for high-throughput sequencing, differently expressed circular RNAs were screened, and GO and KEGG enrichment analyses were performed. The bone marrow single-cell RNA sequencing dataset GSE271107 was obtained from the GEO database. The data of four multiple myeloma bone disease patients were integrated. After quality control and filtering, the batch effect was removed by Harmony method. Subgroup clustering was performed by UMAP, and cell groups were manually corrected after automatic annotation by SingleR. CellChat was used to infer and visualize the intercellular communication in single-cell RNA sequencing data, and analyze the interaction between ligands and receptors.
RESULTS AND CONCLUSION: (1) Compared with healthy controls, the expression levels of 1 265 circular RNAs in peripheral blood exosomes of multiple myeloma bone disease patients were significantly upregulated. (2) GO and KEGG analyses suggested that the parent genes of differently expressed circular RNAs were mainly enriched in pathways in nervous system development, pathways in cancer, PI3K-Akt signaling pathway, Hippo signaling pathway, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, etc., which were closely related to the proliferation, adhesion, migration, and homing of myeloma cells, and multiple myeloma-related complications (such as multiple myeloma bone disease, peripheral neuropathy, and cardiac events). (3) The parent genes of multiple myeloma bone disease-related differential circular RNAs were mainly derived from T cells/natural killer cells, B cells and monocytes/macrophages in the bone marrow microenvironment, which affected osteoclast function by regulating the secretion of multiple cytokines. (4) Monocytes/macrophages, as osteoclast precursor cells, interacted with tumor cells and other immune cells. The MIF pathway was the main pathway involved. The above data preliminarily identified the source cells of multiple myeloma bone disease-related peripheral blood exosomal circular RNA in the bone marrow microenvironment, and found that the ligand-receptor interaction of the MIF pathway between monocytes/macrophages and tumor cells and immune cells may be an important factor in the occurrence of multiple myeloma bone disease.

Key words: multiple myeloma bone disease, exosome, circular RNA, single-cell RNA sequencing, bone marrow microenvironment, osteoclast, PI3K-Akt signaling pathway, Hippo signaling pathway, engineered exosome