Molecular Mechanisms Of MicroRNA-206 In Steroid-induced Avascular Necrosis Of Femoral Head And Roles Of Dixdc1b Gene On Skeletal Development

Steroid-induced avascular necrosis of femoral head(SANFH)refers to the femoral head necrosis caused by insufficient blood supply due to long-term over-dosage administration of hormone drugs such as dehydrocortisol,which remains a serious medical problem associated with clinical use of hormone drugs.PDCD4,as a tumor suppressor,has been found to be a key regulator of apoptosis in tumor and cardiovascular cells,and its expression is regulated by multiple microRNA molecules.Also,PDCD4 is reportedly involved in the osteoclastogenesis process,but its functions,as well as the microRNAs responsible for regulating PDCD4 expression,in pathological processes of steroid-induced avascular necrosis of femoral head still remains unknown.Previous reports showed that miR-206 regulates osteogenesis processes,and its expression was also reported to be increased in animal model of steroid-induced avascular necrosis of femoral head.However,little is known about the pathological roles of miR-206 in steroid-induced avascular necrosis of femoral head,as well as the molecular mechanisms.To investigate the possible roles and interaction of miR-206 and PDCD4 in pathogeneisis of steroid-induced avascular necrosis of femoral head,we carried out a comprehensive study on the functions and mechanisms of miR-206 and PDCD4 expression,as well as their regulatory relationship,in steroid-induced avascular necrosis of femoral head pathogenesis in this study.In clinical tissue samples,we found that expressional levels of miR-206 in gone tissues collected from steroid-induced avascular necrosis of femoral head patients were greatly increased,but expression of PDCD4 gene was significantly down-regulated in clinical bone tissues.To explore the molecular mechanisms of PDCD4 gene expression suppression,we performed a dual luciferase reporter gene assay and observed that miR-206 could directly bind with promoter region of PDCD4 gene and repress its expression in 293T cells.Moreover,we demonstrated that transfection with miR-206 mimics could promote osteoblast apoptosis,suppresse their viability and proliferation capacity,and regulate expression of ALP and apoptosis-regulating proteins Bax and Bcl-2.The transfection with miR-206 inhibitor produced completely contrary results in osteoblasts.Finally,we confired that the apoptosis levels in osteoblasts co-transfected with PDCD siRNA and miR-206 inhibitor were markedly higher than those transfected with only the miR-206 inhibitor,accompanied with corresponding expressional changes in downstream genes ALP,Bax and Bcl-2,indicating that PDCD4 and related signaling pathways mediated the pro-apoptotic role of miR-206 in osteoblasts.To summarize,through assays using clinical tissue samples and osteoblasts in this study,we proved that miR-206 promotes osteoblast apoptosis by suppressing PDCD4 gene expression and regulating its downstream signaling pathoways,thus involved in pathological processes of steroid-induced avascular necrosis of femoral head.Moreover,we performed a preliminary investigation of dixdc1b gene in zebrafish embryo and bone development,revealed that dixdc1b silencing led to serious defects in zebrafish embryo and bone development,showing the key roles of this gene in skeletal development.However,the possible mechanisms of dixdclb gene expression by miRNAs and the specific mechanisms of dixdc1b gene in skeletal development deserve further exploration.