Regulation Of Osteoblast Differentiation And Bone Homeostasis By MiR-196b-5p And The Mechanism Involved

Objective The "imbalance" in the differentiation of bone marrow mesenchymal stem cells(BMSCs)into osteoblasts and adipocytes is closely related to osteoporosis,obesity and other diseases.More and more studies have shown that micro RNA(miRNA)is involved in the regulation of osteogenic and adipogenic differentiation of BMSCs,and affects bone formation,but effects of miR-196b-5p on bone formation has not been reported.Our colleagues have found that miR-196b-5p could inhibit the osteogenic differentiation of BMSCs and induce their adipogenic differentiation in vitro.The purpose of this study was to explore effects of miR-196b-5p on bone formation and reconstruction in vivo by constructing miR-196b-5p transgenic mice in which miR-196 b was overexpressed under the control of the 2.3 kb Col1a1 promoter(Col1a1-miR-196 b Tg).Meanwhile,the target gene of miR-196b-5p and involved signal pathway that regulate the osteogenic differentiation of BMSCs were identified.Finally,effects of miR-196b-5p inhibitor on bone loss in postmenopausal osteoporotic mice were investigated.Methods This study included three parts.In the first part,effects of miR-196b-5p on bone formation and bone reconstruction in vivo were studied through comparing Col1a1-miR-196 b Tg and their wild-type littermates.In fact,we did these experiments:(1)Mice were bred in SPF environment to observe the general phenotype.(2)Changes of bone mass and static parameters of tibial metaphysis were analyzed by X-ray and ?CT scanning.(3)Bone trabeculae,adipocytes and cartilage matrix were observed by alcian blue-hematoxylin-Orange G staining.(4)Expression of alkaline phosphatase(ALP)was revealed by immunohistochemistry.(5)The osteogenic progenitor cells were isolated and cultured in vitro to evaluate their ability to differentiate into osteoblasts and adipocytes.(6)The rate of bone mineralization deposition was calculated by labeling experiment of calcein,which represents the rate of bone formation.(7)Serum osteocalcin expression was detected by ELISA to reflect the change of bone turnover rate.(8)Bone absorption was evaluated by tartrate resistant acid phosphatase(TRACP)staining.In the second part,the target gene and involved signal pathway of miR-196b-5p were identified:(1)Firstly,the candidate target gene of miR-196b-5p was predicted and screened by bioinformatics method and literature review.Then,the candidate target gene was validated by luciferase reporter assay and Western blotting.(2)Regulation of the validated target gene on the osteogenic/lipogenic differentiation of BMSCs were investigated by gain and loss function studies.(3)The relationship between miR-196b-5p and validated target gene on osteogenic and adipogenic differentiation of BMSCs was verified by rescue experiment.(4)The function of miR-196b-5p and validated target gene on osteogenic involved signaling pathway were detected by Western blotting.In the third part of this study,effects of miR-196b-5p inhibitor on bone loss in postmenopausal osteoporotic mice was investigated:(1)miR-196b-5p expression in bone tissue of older mice and in bone marrow stromal cells of OVX mice were detected.(2)miR-196b-5p inhibitor was injected into tibial bone marrow cavity of ovariectomized mice to investigated whether miR-196b-5p inhibitor can reverse the phenotype of bone loss by ?CT and hematoxylin-eosin(HE)staining.Results In the first part,compared with wild-type littermates,Col1a1-miR-196 b transgenic mice showed these changes:(1)significantly shorter at the age of 6 months;(2)Bone Mineral Content/ Bone Volume(BMC/ BV),bone volume / tissue volume(BV/TV),and bone trabecular number(Tb.N)of trabecular in tibial epiphysis and lumbar spine decreased;the structure model index(SMI)and trabecular separation / spacing(Tb.Sp),tibial cortical bone density and cortical thickness(Cortical Thickness,Cort.Th)decreased;(3)Histological experiments show that metaphyseal trabecular bone decreased,number of adipocytes increased and no change in cartilage matrix;(4)osteoblasts which expressed ALP in tibial metaphysis decreased;(5)osteogenic precursor cells showed decreased ability to differentiate into osteoblasts in vitro,and enhanced ability to differentiate into adipocytes;(6)rate of bone mineralization deposition decreased;(7)serum osteocalcin level decreased;(8)the number of osteoclasts decreased.In the second part:(1)Semaphorin 3a(Sema3a)predicted by bioinformatic methods was validated as a target gene of miR-196b-5p by luciferase reporter assay and Western blotting.(2)Overexpression of SEMA3 A induced the osteogenic differentiation of BMSCs and restrained their adipogenic differentiation.Conversely,knockdown of SEMA3 A suppressed osteogenic differentiation and promote adipogenic differentiation of the progenitor cells.(3)Sema3a reversed the regulation of miR-196b-5p on osteogenic/adipogenic differentiation of BMSCs.(4)miR-196b-5p mimics inhibited Wnt/?-catenin signaling pathway,while miR-196b-5p inhibitor and SEMA3 A activated Wnt/?-catenin signaling pathway.In the third part:(1)The expression of miR-196b-5p in cranial tissue of 18-month-old mice was significantly higher than that of 3-month-old mice and higher in bone marrow stromal cells of OVX mice than that of WT mice.(2)After injection of miR-196b-5p inhibitor into tibial marrow cavity of ovariectomized mice,bone mass in tibial metaphysis increased and the number of adipocytes decreased.Conclusion The expression of miR-196b-5p in bone tissue and bone marrow stromal cells is correlated with aging and postmenopausal bone loss.miR-196b-5p can restrain Wnt/?-catenin signaling pathway by block SEMA3 A protein translation.miR-196b-5p abnormal increased in vivo inhibit osteogenic differentiation and bone formation and increase the number of adipocytes,finally lead to osteoporosis.miR-196b-5p inhibitor can increase the bone mass and reduce the number of adipocytes in bone marrow showing its prevention and treatment potential for postmenopausal osteoporosis.In conclusion,this study enriches the understanding about the regulation of bone metabolism and provides a new pathogenic mechanism and prevention target for metabolic bone diseases such as osteoporosis.