P843: aberrant m5c modification of lncrna malat1 is associated with bone lesions formation in multiple myeloma

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: MM is still an incurable disease and osteoclast-mediated bone destruction is a hallmark. Existing agents effectively reduce the number of osteoclasts (OCs) and prevent bone-related diseases but have little effect on overall survival. Investigating the potential mechanisms and developing novel agents against bone lesions is essential. Aims: To investigated the contributions of MM-derived exosomal lncRNAs to osteogenic differentiation and explored through which mechanisms they fulfilled this function and the role of m5C modification in it. Methods: OCs differentiation, bone resorption, bone lesions formation induced by exosomal MALAT1 of MM cells were confirmed by RNA fluorescent in situ hybridization, Tartrate-resistant acid phosphatase (TRAP) staining, von Kossa, and xenograft model in vivo. The mechanism was demonstrated by methylated RNA immunoprecipitation, and RNA stability detection. Results: To explore the possible roles of lncRNAs in bone lesions formation, exosomes were isolated from U266 cells and high‐throughput sequencing was performed. It was found that MALAT1 expression was the highest. Then, MALAT1 expression was examined in MM cohorts. Intriguingly, MALAT1 was significantly increased in MM cells compared with normal plasma (NP) cells in the GSE24080 and GSE5900 patient cohorts (Figure 1A). The FISH examination revealed an obvious abundance of MALAT1 in bone marrow of MM patients (Figure 1B). To demonstrate whether MALAT1 in U266 cells acts on macrophages via incorporating into exosomes, we labeled isolated exosomes with PKH26 dye and MALAT1 with GFP from U266 cells. The labeled exosomes were subsequently added and incubated with RAW264.7 cells. As presented in figure 1C, the majority of the recipient cells exhibited a yellow signal in cytoplasm under the confocal microscope, suggesting that MALAT1 could be packaged into exosomes and swallowed by RAW264.7 cells. We directly influence MALAT1 expression in RAW264.7 cells. Overexpression of MALAT1 could upregulate RANKL expression and increase TRAP-positive OCs and mineralized nodules, while the knockdown of MALAT1 showed a reverse trend (Figure 1D-F). The ability of MM exosomes in bone destruction was then verified in vivo by injecting U266 cells, U266 cells plus MM exosomes, and U266 cells plus MALAT1-knockdown-exosomes into the tail vein of NOG mice. The extent of the bone disease in each model was assessed by micro-CT. Parametric analysis using microCT revealed a significant decrease in the bone volume (bone volume/tissue volume) by exosomes, while when injecting MALAT1-knockdown-exosomes, we observed a reverse trend (Figure 1G). Recent studies focused on the function of RNA modification in regulating lncRNAs expression. We found that MALAT1 level was strongly correlated with NSUN2 (R2=0.758) and YBX1 (R2=0.748) levels (Figure 1H). To test whether and how m5C modification regulates MALAT1 expression, U266 cells were transfected with siRNA targeting NSUN2. Knockdown of NSUN2 significantly reduced the m5C levels of MALAT1 (Figure 1I). A rescue experiment showed that MALAT1 expression and stability increased by NSUN2 were reversed when transfected with YBX1 siRNA (Figure 1J). Summary/Conclusion: Our study demonstrates that myeloma cells-derived exosomal lncRNA MALAT1 might involve in bone lesions formation, which is modulated by NUNS2- mediated m5 C modifications.Keywords: Myeloma, Bone disease, Epigenetic