The Role And Mechanism Of LncRNAgm5532 In Osteoclast Differentiation

Objective:Osteoclasts,derived from the monocyte/macrophage hematopoietic lineage,are responsible for bone resorption which is a process balanced by bone formation to ensure the continual renewal of the skeletal system.Many bone metabolism diseases,such as osteoporosis and rheumatoid arthritis,are associated with excessive osteoclastogenesis.Recent studies have shown that long non-coding RNAs(lncRNAs)play important roles in cellular proliferation,differentiation,and apotosis.However,how IncRNAs involved in osteoclast differentiation is still unclear.Therefore,this study will provide theoretical bases and potential therapeutic targets by revealing the mechanism underlying the differentiation of osteoclast regulated by specific lncRNAs.Methods:(1)Microarrays were performed to profile the expression of lncRNAs and mRNAs during RANKL-induced osteoclastogenesis in RAW264.7 cells.The cluster analysis,GO analysis and Pathway analysis were used to determine the biological functions or pathways of osteoclastogenesis-associated genes.(2)Bioinformatics were performed to screen lncRNAs and the expression of.lncRNA and osteoclastogenesis-associated mRNA was verified by qPCR.(3)siRNA was transfected in bone marrow mononuclear/macrophages to inhibit the expression of lncgm5532,TRAP stain was used to distinguish the osteoclasts and immunofluorescence assay was performed to observe the actin cytoskeleton.Pit formation assay was performed to test the bone resorption of osteoclast,cck8 assay was performed to test the pre-osteoclast cell viability and,finally,western blotting was used to test the protein level of MAPK pathway.(4)Bone marrow mononuclear/macrophages were irradiated by X-ray,TRAP stain was used to distinguish the osteoclasts and the expression of lncgm5532 was examined by qPCR.Results:The microarray assay revealed that lncgm5532 was significantly up-regulated during osteoclast differentiation and the results was further verified by RT-qPCR.After knocking down lncgm5532,osteoclast differentiation and bone resorption were inhibited.The expression level of osteoclast differentiation-related genes and tartrate-resistant acid phosphatase activity were found to be decreased,and the structure of osteoclast actin ring was damaged.Furthermore the proliferation of osteoclasts was inhibited after knockdown of lncgm5532,while the protein expression levels of AKT and ERK decreased.The expression level of XPR1 significantly increased in osteoclastogenesis,whereas the expression of XPR1 mRNA decreased after knockdown of lncgm5532.Conclusion:lncgm5532 regulate osteoclast formation,bone resorption function,and differentiation-related gene expression.lncgm5532 regulates pre-osteoclast viability,the protein level of AKT and ERK,the actin cytoskeleton of osteoclast,and the mRNA expression of XPR1.X-rays irradiation promotes the differentiation of osteoclasts and increases the expression of lncgm5532.