| Osteoporosis（OP）is a common metabolic bone disease syndrome.Its main characteristics are bone loss and bone microstructural destruction,which leads to decreased strength and fragility of bone tissue,which greatly increases the incidence rate of fractures.Menopause Osteoporosis,also known as type OP,occurs in women after menopause for about 5 years.The most common postmenopausal women aged 55-70 years old is more than 200 million people worldwide with osteoporosis.Of them,over 88 million people in China have incidence rate of seventh[1].Its pathogenesis is mainly based on the decrease of hormone level in postmenopausal women,which leads to the breaking of the balance of bone resorption formation between osteoclasts and osteoblasts,further leads to the change of bone tissue structure and the decrease of bone mass,and finally increases the brittleness of bone tissue,resulting in the occurrence of fracture.About 50%of postmenopausal women will have fractures caused by osteoporosis.Fractures can lead to pain,decreased activity and motor function at the onset site,thrombosis and other complications,resulting in increased mortality.Among them,epidemiological studies in North America show that the risk of osteoporotic fractures in various parts of white women over the age of 50 is:distal forearm fracture 16.9%,hip fracture 17.5%,vertebral fracture 15.6%,which is much higher than the corresponding fracture risk of men of the same age,which are 3.0%,6.0%and 5.0%respectively[2].The significantly increased incidence of fracture（especially hip fracture）is easy to cause various economic and life burdens to the patients themselves,their families and society.At the same time,the prognosis is poor and the disability rate is high,which seriously affects the physical and mental health of elderly women.Therefore,the prevention and treatment of postmenopausal osteoporosis（OPM）and its associated fractures is one of the hotspots of clinical research in internal medicine and orthopedics in recent years.At present,there are many methods for clinical treatment of OPM,including new drugs such as diosuMab,and other methods including bisphosphonate,hormone replacement,calciuM supplementation,bone resorption,etc.but because of the many basic diseases,poor compliance,adverse effects of drug and unstable efficacy,the treatment effect is not satisfactory,and the incidence rate of osteoporosis related fractures has not been effectively controlled.Bone marrow mesenchymal stem cells are adult stem cells derived from mesoderm.Their main characteristics are self-renewal and multidirectional differentiation potential.They can differentiate into various cells and play an important role in the development,regeneration and repair of bone tissue.Osteoblasts and adipocytes are differentiated from BMMSCs,which dynamically affects the dynamic balance of osteoblast differentiation and the steady-state balance of bone tissue remodeling.Existing studies have shown that the important reason for osteoporosis is that during the occurrence of osteoporosis,the osteogenic differentiation ability of BMMSCs decreases and the adipogenic differentiation ability increases[4].The abnormal differentiation of BMMSCs directly affects the development,regeneration and repair of bone tissue,but the relevant mechanism of how to regulate the multidirectional differentiation of BMMSCs is unclear.MicroRNA（miRNA）is a newly discovered non coding microRNA,which widely exists in animals and plants.They function by binding to the mrna3’-untranslated region of the target gene mainly through base pairing,so as to inhibit or silence the expression of the target gene at the post transcriptional level,and play an important regulatory role in a variety of physiological and pathological processes.It has been found that a variety of miRNAs are involved in the osteogenic adipogenic differentiation of stem cells and play an important role in regulating their fate.In recent years,the research on miRNA has always been a hot spot of attention.With the gradual deepening of understanding,more than 1000 kinds of miRNA have been found in various animals,plants and viruses.At the same time,it is found that miRNA plays an important role in various biological activities,including cell proliferation and differentiation,cell death and apoptosis,lipid metabolism,bone tissue reconstruction and even tumor development and invasion,are regulated by various miRNAs.However,it is not clear which miRNAs play a key role in the multidirectional differentiation of BMMSCs,which miRNAs regulate the function of BMMSCs through those molecular signal pathways,and whether miRNAs play a role in the formation and development of OPM.There is still no specific research to explain the above problems.The current view is that the destruction of the balance between the original formation and absorption of bone tissue caused by the decline of estrogen level is the root cause of postmenopausal osteoporosis,which leads to the disorder of bone tissue reconstruction,the increase of bone loss and brittleness,and then increases the risk of various fractures.Previous studies mainly focused on the abnormal increase of bone resorption mediated by abnormal activation of osteoclasts,while there were few studies on the decrease of osteoblast mediated bone formation.Bone marrow mesenchymal stem cells are pluripotent stem cells and ideal seed cells for tissue engineering.They are not only the source cells of osteoblasts in bone tissue,but also the source cells of adipocytes.The number and function of osteoblasts are directly affected by the number and function of bone marrow mesenchymal stem cells.Existing studies have shown that the root cause of postmenopausal osteoporosis is the abnormal differentiation of bone marrow mesenchymal stem cells,mainly the decrease of osteogenic differentiation ability and the increase of adipogenic differentiation ability,resulting in the decrease of the number of osteoblasts in bone marrow and the increase of the number of adipocytes[4],but the molecular mechanism of the imbalance of bone marrow mesenchymal stem cell differentiation is still unclear.The osteogenic adipogenic differentiation process of bone marrow mesenchymal stem cells is relatively complex,which is regulated by a variety of cell molecular signals and multiple pathways,and finally leads to their normal or abnormal differentiation.A variety of transcription factors and multiple signaling pathways play a regulatory role in the whole process.Therefore,in this experiment,we first established the osteoporosis mouse model and compared two kinds of BMMSCs isolated and purified from osteoporosis model mice and normal mice.According to relevant studies,miR-181c-5p of the two sources of BMMSCs was differentially expressed between normal mice and osteoporosis model mice after gene screening[3]Therefore,miR-181c-5p was selected for further functional study in this experiment.In this experiment,we selected normal bone marrow mesenchymal stem cells and abnormal bone marrow mesenchymal stern cells extracted from osteoporosis model,which had differential expression of miR-181c-5p.From the target level,we discussed its effect and mechanism on the abnormal differentiation of bone marrow mesenchymal stem cells in the pathogenesis of postmenopausal osteoporosis,so as to further deepen the understanding of the pathogenesis of postmenopausal osteoporosis,At the same time,the root cause of bone loss in postmenopausal osteoporosis was clarified.Finally,this experiment provides an important guiding role for the prevention and treatment of fracture diseases caused by postmenopausal osteoporosis,and provides a new direction and target for the drug research and development of postmenopausal osteoporosis.Objective:（1）To explore which miRNAs are differentially expressed between bone marrow mesenchymal stem cells derived from osteoporosis and normal bone marrow mesenchymal stem cells,select miR-181c-5p to further discuss the regulatory role of its bone marrow mesenchymal stem cells in the whole process of osteogenic adipogenic differentiation,and determine its target genes and related mechanisms.（2）To investigate whether the abnormal osteogenic adipogenic differentiation of bone marrow mesenchymal stem cells derived from osteoporosis can be reversed after restoring the expression level of differentially expressed mr-181c-5p in bone marrow mesenchymal stem cells.Methods:（1）The mouse model of postmenopausal osteoporosis（OPM）was established and successfully established by ovariectomy.Two kinds of bone marrow mesenchymal stem cells（O-BMMSCs）derived from osteoporosis and bone marrow mesenchymal stem cells（S-BMMSCs）derived from sham operation were successfully established by ovariectomy.Finally,miR-181c-5p with differential expression between them was screened for further study.（2）The expression level of miR-181c-5p in bone marrow mesenchymal stem cells was changed by cell transfection related technology.After overexpression or inhibition of miR-181c-5p,alizarin red staining,oil red O staining,MTT analysis,qRT-PCR and western blot related technology were used to further detect the effect of different expression levels of miR-181c-5p on the proliferation of bone marrow mesenchymal stem cells and the regulation of osteogenic adipogenic differentiation ability of bone marrow mesenchymal stem cells.The candidate target gene FoxO1 of miR-181c-5p was screened by target gene prediction software,and the function of target gene FoxO1 was verified.（3）Down regulate the expression level of miR-181c-5p in O-BMMSCs.After cell transfection,alizarin red staining,oil red O staining,qRT-PCR and western blot were used to further analyze whether it can reverse the abnormal osteogenic adipogenic differentiation of O-BMMSCs.Results:（1）Overexpression of miR-181c-5p and inhibition of miR-181c-5p after cell transfection had no significant effect on the proliferation of bone marrow mesenchymal stem cells.After transfection,up regulating the expression of miR-181c-5p in bone marrow mesenchymal stem cells can reduce the osteogenic ability of bone marrow mesenchymal stem cells and enhance the adipogenic ability of bone marrow mesenchymal stem cells,while down regulating the expression of miR-181c-5p in bone marrow mesenchymal stem cells can increase the osteogenic ability of bone marrow mesenchymal stem cells and inhibit the adipogenic ability of bone marrow mesenchymal stem cells.The target gene prediction software predicts that the candidate target of miR-181c-5p is FoxO1.Up regulating the expression of miR-181c-5p in bone marrow mesenchymal stem cells by transfection can lead to the down regulation of FoxO1 protein expression,while down regulating the expression of miR-181c-5p in bone marrow mesenchymal stem cells can lead to the up regulation of FoxO1 protein expression.Luciferase Report can further prove that miR-181c-5p can specifically bind to FoxO1.（2）After cell transfection,the expression of miR-181c-5p in O-BMMSCs was inhibited,the protein expression of FoxO1 was up-regulated,the osteogenic differentiation ability of O-BMMSCs was improved,the abnormal adipogenic differentiation ability of O-BMMSCs was reduced,and finally the normal differentiation ability of O-BMMSCs was partially restored.Conclusion:（1）MiR-181c-5p further regulates the osteogenic adipogenic differentiation of bone marrow mesenchymal stem cells by negatively regulating the expression of target gene FoxO1.By down regulating the overexpression of miR-181c-5p in O-BMMSCs;its osteogenic adipogenic differentiation was finally partially reversed.（2）The overexpression of miR-181c-5p exists in the occurrence of osteoporosis,which destroys the balance of osteogenic adipogenic differentiation in bone marrow mesenchymal stem cells and reduces the bone formation ability of bone marrow mesenchymal stem cells.It is one of the reasons for the decrease of bone mass and bone mass in whole body bone tissue. |
