Effects Of ANCR On Osteoclast Differentiation And Its Mechanism

Objective:Osteoporosis is a metabolic bone disease characterized by decreased bone mass and microstructure destruction of bone tissue,resulting in increased bone fragility and easy fracture.The main reason is that excessive bone resorption exceeds the bone formation of osteoblasts,which leads to osteoporosis.Previous studies have shown that lnc RNA is closely related to the formation of osteoclasts.This study mainly explores the role of Lnc RNA-ANCR in the process of osteoclast differentiation and its possible mechanism,and provides a possible theoretical basis for studying the formation and differentiation mechanism of osteoclasts.Provide a new target and basis for the treatment of osteoporosis.Methods:Extract C57BL/6 mouse bone marrow primary mononuclear cells in macrophage colony-stimulating factor and the receptor activator of NF-κB ligand stimulates the differentiation of macrophages and macrophages into osteoclasts.Phalloidin,Tartrate-resistant acid phosphatase staining and TRAP enzyme activity were used to verify the formation of osteoclasts.In situ hybridization was used to detect and compare the expression of ANCR during the differentiation of bone marrow-derived macrophages into osteoclasts.This study also compared BMMs and macrophage cell line RAW264.7cells in the differentiation of osteoclasts under the stimulation of M-CSF and RANKL.Constructed ANCR overexpression lentivirus,and explored the effect of ANCR overexpression lentivirus on the differentiation of RAW264.7 cells into osteoclasts.The concentration of the virus was1×10~9TU/ml.The overexpression ANCR lentivirus and the negative lentivirus were infected with RAW264.7 respectively.Cells were obtained after stably transfected cells were induced and cultured,and fluorescent quantitative PCR was used to verify the transfection efficiency of ANCR.The RAW264.7 cells were randomly divided into 4 groups:control group,induction group,induction+lentivirus ANCR group,induction+negative lentivirus group.Except for the control group,the cells of the other 3groups were induced withα-MEM complete medium containing 50ng/ml M-CSF with 100ng/ml RANKL.The control group was cultured withα-MEM complete medium for 6 days.Phalloidin staining to observe whether there are osteoclasts and the proportion of osteoclasts to the total cells counted.Use SPSS,Image J software for measurement statistics,Graphpad Prism7.0 for data mapping.Results:Primary BMMs and macrophage cell line RAW264.7 cells were cultured with 50ng/ml M-CSF and 100ng/ml RANKLα-MEM complete medium to induce osteoclasts,but RAW264.7 cells have the characteristics of being more easily induced into osteoclasts.In situ hybridization data showed that with the extension of the induction time,the expression of ANCR decreased significantly,and reached the lowest level on the 6th day.The difference in the results of this study was statistically significant(P<0.05).Fluorescence quantitative PCR results discovery that contrasted with the blank group,there is no statistically significant difference in the expression of ANCR between the ANCR overexpression lentivirus group and the negative lentivirus group(P>0.05).There wasn’t significant difference in the positive rate of osteoclasts among the induction group,ANCR overexpression lentivirus+induction group,and negative lentivirus+induction group,and the difference in the result haven’t statistically significant(P>0.05).Conclusion:Primary BMMs and RAW264.7 cells can be successfully induced into osteoclasts by M-CSF+RANKL stimulation.ANCR may play a negative regulatory role in the process of osteoclast differentiation,which provides a new prevention strategy for the occurrence of osteoporosis.