[The Mechanism of Multiple Myeloma U266 Cell Exosome Circ- G042080 Promoting Multiple Myeloma Bone Disease through MiR-6791-3p/HDAC4 Axis].

OBJECTIVE

To explore the mechanism of multiple myeloma cell exosome circRNA inducing osteoclast differentiation and promoting myeloma bone disease, and explore the feasibility of intervening myeloma bone disease by regulating circRNA-mediated cell communication.

METHODS

TargetScan, miRanda and dual luciferase reporter assays were used to predict and verify the downstream regulatory mechanism of circ-G042080. The exosomes of myeloma U266 cells were identified by electron microscopy. RANKL induced RAW264.7 cells to differentiate into osteoclasts. By co-culturing with U266 cells with or without exosomes, TRAP staining, Von Kossa staining and Western blot were used to determine the effect of U266 cell exosomes on osteoclast differentiation. The human myeloma xenograft model was constructed by injecting U266 cells into the tail vein of NOG mice. The effect of myeloma-derived exosome circ-G042080 on bone destruction was evaluated by micro-CT, immunohistochemistry and immunofluorescence. By knocking down or overexpressing circ-G042080, the mechanism of osteoclast differentiation induced by regulating miR-6791-3p/HDAC4 was further clarified.

RESULTS

Bioinformatics analysis and dual luciferase reporter assay found and verified that circ-G042080 could competitively bind to miR-6791-3p through the ceRNA mechanism to regulate HDAC4 expression. RANKL successfully induced RAW264.7 cells to differentiate into osteoclasts. After co-cultured with U266 cell exosomes, the number of TRAP and Von Kossa positive cells increased significantly (both P < 0.01), and the expression of HDAC4 and RANKL protein increased significantly (both P < 0.01), suggesting that U266 cell exosomes promoted osteoclast differentiation. Compared with the control group, the mice in the model group showed obvious bone destruction, increased number of TRAP-positive osteoclasts ( P < 0.01), and significantly increased expression levels of HDAC4 and RANKL proteins (both P < 0.05). At the same time, immunofluorescence showed that the expression of circ-G042080 was significantly increased ( P < 0.01). After injection of U266 cell exosomes, the bone destruction of femur, spine and skull in mice was significantly aggravated compared with the model group, and the levels of circ-G042080, HDAC4 and RANKL were significantly increased (all P < 0.01). Finally, by knocking down or overexpressing circ-G042080, it was confirmed that it regulated the miR-6791-3p/HDAC4 axis, promoted RANKL expression, and then induced osteoclast differentiation.

CONCLUSION

Myeloma exosome circ-G042080 can promote RANKL-mediated osteoclast differentiation through the miR-6791-3p/HDAC4 axis and promote myeloma bone disease, which is expected to become a new biomarker.