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.
题目: 多发性骨髓瘤U266细胞外泌体circ-G042080通过miR-6791-3p/HDAC4轴促进骨髓瘤骨病的机制研究.
目的: 探究多发性骨髓瘤细胞外泌体circRNA诱导破骨细胞分化、促进骨髓瘤骨病的作用机制，并探讨通过调控circRNA介导的细胞通讯干预骨髓瘤骨病的可行性。.
方法: TargetScan、miRanda及双荧光素酶报告实验预测并验证circ-G042080下游调控机制。应用电镜鉴定骨髓瘤U266细胞外泌体，RANKL诱导RAW264.7细胞分化为破骨细胞，通过将其与U266细胞伴或不伴外泌体共培养，借助TRAP染色、Von Kossa染色和Western blot明确U266细胞外泌体对破骨细胞分化的作用。通过对NOG小鼠尾静脉注射U266细胞构建人源性骨髓瘤移植瘤模型，应用micro-CT、免疫组化、免疫荧光评价骨髓瘤来源外泌体circ-G042080对骨破坏的作用。通过敲减或过表达circ-G042080，进一步明确其通过调控miR-6791-3p/HDAC4诱导破骨细胞分化的作用机制。.
结果: 生信分析及双荧光素酶报告实验发现并验证了circ-G042080能够通过ceRNA机制，竞争性结合miR-6791-3p，以调控HDAC4表达。RANKL成功诱导RAW264.7细胞分化为破骨细胞，与U266细胞外泌体共培养后，TRAP、Von Kossa阳性细胞数显著增加（均 P < 0.01），HDAC4、RANKL蛋白表达显著升高（均 P < 0.01），提示U266细胞外泌体对破骨细胞分化具有促进作用。与对照组小鼠相比，模型组小鼠表现出明显的骨质破坏、TRAP染色阳性的破骨细胞数目增加（ P < 0.01），HDAC4、RANKL蛋白表达水平显著升高（均 P < 0.05），同时免疫荧光显示circ-G042080表达显著提高（ P < 0.01）。注射U266细胞外泌体后，小鼠股骨、脊柱、颅骨骨质破坏较模型组显著加重，circ-G042080及HDAC4、RANKL水平显著升高（均 P < 0.01）。最 后，通过敲减或过表达circ-G042080证实了其调控miR-6791-3p/HDAC4轴，促进RANKL表达，进而诱导破骨细胞 分化。.
结论: 骨髓瘤外泌体circ-G042080可通过miR-6791-3p/HDAC4轴促进RANKL介导的破骨细胞分化，促进骨髓瘤骨病，有望成为新型生物标志物。.
Keywords: multiple myeloma bone disease; exosome; circ-G042080; histone deacetylase 4; receptor activator for nuclear factor κB ligand; osteoclast differentiation.