MiR-21Targeting Sprouty1Regulates Osteoblast Differentiation Of Human Mesenchymal Stem Cell And Contributes To Postmenopausal Osteoporosis

In chronic inflammatory related bone disease, attenuated bone formation isa key mechanism in the pathology of these diseases. Mesenchymal stem cells(MSCs) are known as progenitor cells of osteoblasts and osteocytes in boneformation. Studies have shown a reduction in the differentiation of MSCs intoosteoblasts contributes to the impaired bone formation observed in osteoporosis.Therefore, investigatingthe molecular mechanisms that regulate human bonemarrow mesenchymal stem cell (hBMMSC) differentiation intoosteogeniclineage is important for the development of anabolic therapiesfortreatment of osteoporosis.MicroRNA (miRNA) function at the posttranscriptionallevel throughnegative regulation their target genes. MiRNA have shown played an important role in regulating a variety of physiological and pathological processes,including cell differentiation, proliferation, and cancer. It has indicated thatmultiple miRNAs could regulate osteogenesis in different cellular context. Buthow to identify key miRNAs involved in screening in osteogenesis ofhBMMSCs, what is the mechanism of these miRNAs to regulate hBMMSCsosteogenic differentiation, and whether the abnormal expression and functionof these miRNAs contributes to the pathogenesis of postmenopausalosteoporosis are still unclear.In previous study we screened differentially expressed miRNAs inosteogenesis of tissue-specific mesenchymal stem cells (hPDLSCs). In order toconfirm these miRNAs also played a role in hBMMSC osteoblastic differentiationwe searched the microarray results of hBMMSCs osteogenesis and finally focusedon miR-21which was previously reported up-regulated during osteogenesis ofhBMMSCs. Studing the regulatory role of miR-21in hBMMSCs osteogenicdifferentiation as well as abnormal expression and function of miR-21inpostmenopausal osteoporosis would help us to understand the mechanism ofosteogenic differentiation and develop promising stragegies to treat osteoporosis.Objectives1. To investigate the expression of miR-21during osteogenic differentiationof hBMMSCs.2. To study the role of miR-21on osteogenic differentiation of hBMMSCs invitro and in vivo.3. To explore the molecular mechanism of miR-21in regulating osteogenesisof hBMMSCs.4. To investigate the expression and function of miR-21in hBMMSCsderived from postmenopausal osteoporosis (PMOP-hBMMSCs). Methods1. The improved density gradient centrifugation combined with whole bonemarrow adherence on plastic to isolate and culture human bone marrowmesenchymal stem cells. Phenotypes of hBMMSCs were analyzed byflow cytometry analysis. hBMMSCs proliferation potential determinedby colony formation and MTT. Cell cycle analysis was determined byflow cytometry.The multi-differentiation potential of hBMMSCs wasassayed by osteogenic, adiopogenic and neurogenic differentitation.Alizarin Red S, Oil red O and immunofluorescence staining was used todetermine the differentiation ability. And osteogenic and adipogenicspecific genes were determined by real time RT-PCR.2. miR-21was over-expressed or inhibited using synthetic pre-miR-21oranti-miR21(complementary to mature miR-21), respectively. Theexpression levels of miR-21after transfection were confirmed by realtime RT-PCR. After2days of transfection, hBMMSCs were induced toosteoblast differentiation. ALP and Alizarin red S staining were used todeterminee osteogenic ability. Real time RT-PCR and western blot wereused to analysesestrogenic marker genes expression. To studywhetherup-regulated miR-21enhances in vivo ectopic boneformation,hBMMSCs transfectedwith pre-miR-21, anti-miR-21or miR controlwere loaded on hydroxyapatite-tricalcium phosphate scaffold andimplanted s.c. inNOD/SCID mice for8weeks. HE staining andMasson’s trichromestaining were used to assay the osteoid formation.3. We confimed miR-21targets the3′UTR of Spry1by bioinformaticscombined with luciferase reporter detection and protein analysis. themiR-21target gene Spry1. In order to study the role of Spry1on hBMMSCs osteogenic differentation, the expression of Spry1duringosteogenesis was determined by real time RT-PCR. We also constructlentiviral vector of Spry1to overexpress Spry1in hBMMSCs. ALP,Alizarin red staining and RT-PCR and Western blot analysis were used todetermine the osteogenic potential of hBMMSCs after overexprssionSpry1.4. Real time RT-PCR was used to detect the expression of miR-21inH-hBMMSCS and PMOP-hBMMSCs. Spry1gene and proteinexpression in H-hBMMSCS and PMOP-hBMMSCs were confirmed byreal time RT-PCR and western blot analysis. The osteogenic potentialwas analysed after over-expression miR-21in PMOP-hBMMSCsResults1. Cultured hBMMSCs expressed surface markers of mesenchymal, ratherthan the expression of hematopoietic markers. Clone colony and MTTassay showed that hBMMSCs have the ability of self-renewal andreplication. Osteogenic, adipogenic, chondrogenic induction showed thathBMMSCs could differentiate into multiple mesoderm-type lineages.2. miR-21was incresed during osteogenic differentation of hBMMSCs.Gain-loss-functional analysis showed that up-regulating miR-21couldpromote hBMMSCs osteogenic differentiation in vitro and in vivo. On thecontrary, silencing of miR-21could inhibit the osteogenic differentiationof hBMMSCs in vitro and in vivo.3. In order to further study the molecular mechanisms of miR in regulation ofhBMMSCs osteogenic differentiation. Spouty1(Spry1) was selected bybioinformatics prediction.miR-21was confirmed to target Spry13＇UTRbyluciferase report analysis and western blot analysis. Spry1was down-regulated during hBMMSCs osteogenic differentation, which wasopposite to the expression of miR-21. Spry1function analysis found thatthe increased Spry1could attenuated the osteogenic differentiation ofhBMMSCs.4. The osteogenic differentiation was impaired in hBMMSCs derived frompost-menopausal osteoporosis. Furthermore,the expression of miR-21wasdecreased while Spry1was increased in PMOP-hBMMSCs. Up-regulatingmiR-21could partially restore the impaired osteogenic potential inPMOP-hBMMSCs with reduced Spry1expression.Conclusion1. miR-21could promote osteogenic differentiation of hBMMSCs in vitro.2. miR-21could enhanceectopic Bone Formation in vivo.3. miR-21target Spry13＇UTR and Spry1negatively regulated osteogenicdifferentiation of hBMMSCs.4. miR-21was decreased while Spry1was increased in PMOP-hBMMSCs.Over-expression of miR-21could partially rescued the impairedosteogenic ability of PMOP-hBMMSCs with down-regulated Spry1.