ER?-SATB2 Pathway Regulates Stemness And Osteogenesis Of Bone Marrow Stromal Cells To Preventpostmenopausal Osteoporosis

Objective:To study the molecular mechanism ofER?-SATB2 pathwayregulating stemness and osteogenesis of bone marrow stromal cells to prevent postmenopausal osteoporosis and to provide a new theoretical and experimental basis for the treatment of postmenopausal osteoporosis.Methods:(1).The ovariectomized rat model of postmenopausal osteoporosis was established.BMSCs were isolated and cultured from bonesof both ovariectomized and sham rats to compare their biological characters.Micro-CT and Hematoxylin-eosin(HE)staining were used to compare the histomorphology of limbs in two groups.The stemness of the two groups BMSCs was analyzed by Colony-forming assays,RT-PCR and Western blot.After induction of osteogenic differentiation,alizarin red staining was used to detect the formation of calcium nodules.RT-PCR and Western blot were used to analyze the expressions of related osteogenic factors.(2).Three groups of Sham-BMSCs,OVX-BMSCs,OVX-BMSCs+estrogen(E2)were designed.The clonal formation rate,the expression of stemness factor,the formation of calcium nodules and the changes of osteogenesis-related genes were analyzed.The expression of SATB2 was also observed.(3).OVX-BMSCs were added with siRNA-ER?,ICI 182,780(estrogen receptor inhibitor),respectively.The clonal formation rate,the expression of sternness factor,the formation of calcium nodules and the changes of osteogenesis-related genes were analyzed.The expression of SATB2 was also observed.(4).Bioinformatics software predicted the potential ERE sequence of the upstream promoter region of SATB2.The binding sites of ER? in the SATB2's promoter region were verified by chromatin immunoprecipitation and dual luciferase reporter assay.(5).Construction of SATB2 overexpression plasmids.After transfection of OVX-BMSCs,the dryness and osteogenic differentiation ability were analyzed.BMSCs overexpressing SATB2 were injected into the OPM animal model by tail vein,and then the bone tissue of the limbs of the two groups was analyzed by Micro-CT and HE staining Morphological analysis.Results:(1).The OPM rat animal model was successfully established.Micro-CT and HE staining showed that bone mineral density,bone trabecula number and volume fraction of limbs in Sham rats were higher than those in OVX group.The clonal formation rate of Sham-BMSCs and the expression of Nanog,Sox2 and OCT4 were higher than those of OVX-BMSCs,which were statistically significant.After induction of osteogenic differentiation,the expressions of Runx2 and OCN in Sham group were significantly higher than that in OVX group(P<0.05).(2).OVX-BMSCs group was treated with 10-8M estrogen and cultured for 24 hours.Compared with Sham-BMSCs and OVX-BMSCs,the stemness and osteogenic differentiation of OVX-BMSCs were higher than those of OVX-BMSCs,and the expression level of SATB2 was also higher than that of OVX-BMSCs.(3).OVX-BMSCs group were added with siRNA-ER?,ICI 182,780.Compared with the OVX-BMSCs group,the sternness and osteogenic differentiation of BMSCs in siRNA-ER? group decreased,while the decrease in sternness and osteogenic differentiation of BMSCs in ICI182,780 group was consistent with that of siRNA-ERP group.There was no statistically significant difference between the two groups.The expression level of SATB2 was decreased in both siRNA-ER? and ICI182,780 groups.Considering the expression of SATB2 in Sham-BMSCs and OVX-BMSCs,the results showed that the expression of SATB2 was closely related to ER? under the intervention of estrogen.(4).Bioinformatics software screened three ERE sequences in the upstream promoter region of SATB2,and constructed luciferase reporter plasmids to detect the fluorescence ratio(FL/RL),and concluded that sequences A and B were significant.The CHIP experiment further confirmed that ER? binds to the sequence A and B of S ATB2 and regulates its expression.(5).SATB2 virus vector was transfected into OVX-BMSCs.The clonal formation rate of BMSCs increased,and the expression levels of sternness factor Nanog,Sox2 and Oct4 increased as well.The number of osteoblasts was enhanced,and the expression levels of osteogenesis-related genes Runx2 and OCN were up-regulated.(6).BMSCs that overexpressed SATB2 were injected into the OPM animal model by tail vein injection.After 2 months,Micro-CT and HE staining showed that the bone mineral density,the number of trabecular bone and the volume of trabecular bone were larger than those in the control group.Conclusion:Our experiment demonstrated that ER?-SATB2 pathway regulates the stemness and osteogenic differentiation of BMSCs and participates the process of post-menopausal osteoporosis in vivo and in vitro.Gene-modified SATB2 has the feasibility and efficacy to reverse postmenopausal osteoporosis.