MiR-664-3p Promotes Osteoporosis Via Targeting Smad4 And Osterix

Osteoporosis is characterized by microarchitectural deterioration,low bone mass,and increased risk of fractures,and recent trail results indicate that improving impaired bone formation by boosting the function of differentiated or mature osteoblasts may be a new and improved strategies for treating osteoporosis.MicroRNAs(miRNAs)repress cellular protein levels to provide a sophisicated parameter of gene regulation that plays important roles in physiology and diseases,and present tremendous therapeutic potential.miRNAs are dysregulated in osteoporosis but whether they control osteoblast differentiation and skeletal biology or constitute therapeutic targets remains underexplored.Icariin(ICA)is a main active flavonoid glucoside isolated from Herbaepimedii,which has long been used to treat osteoporosis.In this study,using miRNA high-throughput sequencing,we screened the differentially expressed miRNAs between Icariin induced mosue preosteoblast MC3T3-E1 cells and control cells.Partial differentially exoressed miRNAs were further validated by real-time PCR,and miR-664-3p with the highest fold of downregulation and potential targets of Smad4 and Osx was selected to be studied further.Smad4,a key transducer of the osteogenic signal,and Osterix,an early response gene essential for osteoblast differention and bone formation,were demonstrated as target genes of miR-664-3p by luciferase reporter assay,miR-664-3p functionally inhibited the differentiation of mouse preosteoblasts MC3T3 E1 and mesenchymal stem cells C3H10T1/2 by attenuating Smad4 and Osterix pathways that synergistically contribute to bone formation.Overexpression of Smad4 and Osx can rescue the inhibiting osteoblast differentiation by mi R-664-3p.Furthermore,we constructed ovariectomy-induced osteoporosis model of mice and found that mi R-664-3p level was increased in the bone of osteoporosis mice compared to the sham-operated mice.Treated with ICA can attenuate the ovariectomy-induced osteoporosis and elevated miR-664-3p level was inpaired by ICA treatment.Therefore,ICA alleviated bone loss in treating osteoporosis may by inhibit miR-664-3pexpresssion.Putative hsa-miR-664-3p recognition sites in human Smad4 and Osx 3’UTR were predicted by TargetScan Human.Hsa-miR-664-3p functionally inhibited differentiation of human mesenchymal stem cells by inhibiting Smad4 and Osterix expression.Identification of circulating miRNAs as biomarkers of osteoporosis is an important step for establishment of new diagnostic and therapeutic approaches.Using the serum of 26 osteoporotic and 18 nonosteoporotic patients,we found that miR-664-3p in the serum of osteoporotic patients were significantly upregulated compared with that of nonosteoporotic patients.Taken together,miR-664-3p was identified as a critical suppressor of osteoblastogenesisby targeting Smad4 and Osterix.Upregulation of miR-664-3p promoted the occurrence of osteoporosis.miR-664-3p was involved in the prevention of Ovx-induced osteoporosis by ICA treatment.miR-664-3p levels were elevated in the serum of osteoporotic patients,suggesting that it may be used as a biomarker for the diagnosis of osteoporosis.Results of this study also indicated that miR-664-3p may be a target for the prevention and treatment of osteoporosis.