Abstract
Purpose: How exosomic lncRNAs contribute to the development of osteogenic differentiation in the context of the bone marrow microenvironment (BMMe) has not been previously described in multiple myeloma (MM). We hypothesized that the BMMe affects osteogenic differentiation of MSCs through exosomic lncRNAs. We aimed to assess which exosomic lncRNAs are involved and through which molecular mechanisms they elicit the hypothesized function.
Methods: We isolated exosomes and confirmed their identity using transmission electron microscopy, nanoparticle tracking analysis and western blot. Co-culture experiments were performed to assess exosomic transfer of lncRUNX2-AS1 from MM cells to MSCs. RNase protection assay was used to examine the possibility of RNA duplexformation between lncRUNX2-AS1 and RUNX2. To investigate whether the overlapping RNA duplex affects the stability of the mRNA, we blocked any new mRNA synthesis with α-amanitin treatments. We developed in-vivo mouse models of bone marrow-disseminated human myeloma to evaluate bone activities. Student's t test was employed to assess the differences between treatment groups.
Results: lncRNA profile analysis and sequential screening identified highly abundant lncRUNX2-AS1 in MSCs from MM. lncRUNX2-AS1 could be packaged into exosomes and transferred to recipient cells, resulting in the reduction of osteogenic differentiation of MSCs. Furthermore, lncRUNX2-AS1 and RUNX2 formed an RNA duplex and mutually decreased their stability. As a pair of protein-coding cis-sense/non-coding antisense transcripts, RUNX2 and lncRUNX2-AS1 were dysregulated simultaneously and correlated negatively in MSCs, driving osteogenic differentiation of MSCs. Invivo mouse models, intraperitoneally administered GW4869, an inhibitor of exosome secretion, was found to be effective in prevention of bone loss, sustained by both bone-forming and anticatabolic activities.
Conclusions: We identified an antisense lncRUNX2-AS1 formed sense-antisense pairs by pairing with a protein-coding gene RUNX2 on the opposite strand to regulate mRNA stability. Our results indicated a unique role of exosomic lncRUNX2-AS1 in transferring from MM cells to MSCs in osteogenic differentiation, through a novel exosomic lncRUNX2-AS1/RUNX2 signaling pathway.
Figure legend. Characterization of exosomes derived from HMCLs (A) Representative TEM imaging of exosomes derived from U266 and MM1S (Bar = 100 nm). (B)Exosomes isolated from U266 and MM1S were measured by Nanoparticle Tracking Analysis (NanoSight). (C) Representative western blot of HSP70 and flotillin-1 proteins in MSCs-derived exosomes and HMCLs-derived exosomes. (D) BM-MSCs were cultured in the absence (control) or presence of U266 or MM1S-derived PKH67-labeled exosomes for 24 hours. Exosomes were uptaken by BM-MSCs, as shown using a confocal microscope (original magnification, ×400). BM-MSCs were stained using DAPI (nuclei) and FITC conjugated anti-actin antibody. (E) Flow cytometric analysis of HMCLs after incubation with fluorescently labeled exosomes. FL1 fluorescence indicates exosome uptake.
No relevant conflicts of interest to declare.
