TNF-α Suppresses The Mesenchymal Stem Cell Osteogenesis Promoter MiR-21in Estrogen Deficiency-induced Osteoporosis

Estrogen deficiency-induced osteoporosis, which is characterized by animbalance between bone formation and bone resorption, is caused by elevatedinflammatory cytokines, such as TNF-α, IL-1, IL-6, and IFN-γ. Studies haveshown that inflammatory cytokines increase the activity of osteoclasts andactivate bone resorption; therefore, antiresorptive therapy is widely used in themanagement of osteoporosis. This type of treatment, however, only preventsfurther loss of bone while barely stimulates new bone formation and reversesbone loss. Recent studies have shown that increased levels of inflammatorycytokines also impaired bone formation in postmenopausal osteoporosis andother inflammatory bone diseases, such as arthritis and periodontitis. Thus,understanding the mechanism of impaired osteoblastic bone formation byinflammatory cytokines may be crucial for developing new and improvedstrategies for treating osteoporosis and inflammatory bone diseases.Bone Mesenchymal stem cells are known as progenitor cells of osteoblasts andosteocytes in bone formation. A reduction in the differentiation of BMSCs into osteoblasts contributes to the impaired bone formation observed in osteoporosis,and the commitment and differentiation of BMSCs into osteoblasts may beaffected by TNF-α, which plays a central role in the pathogenesis ofpostmenopausal osteoporosis. The responsible mechanism is, however, not wellelucidated. The miRNAs are a newly discovered class of post-transcriptionalregulators, and they play critical roles in regulating BMSCs osteogenicdifferentiation and bone formation. Studies have shown that inflammatorycytokines could regulate miRNAs and contribute to cancer and someinflammatory diseases. In a previous study, a novel mechanism were identified inbone disease in which miRNA was affected by a local inflammatory environment,especially TNF-α, which was closely related to defective osteogenesis oftissue-specific BMSCs. Those findings, however, were only based ontissue-specific BMSCs, and no well-established animal model was used.Based on this, we employed an ovariectomized animal model to further confirmwhether miRNA, which is regulated by a systemic inflammatory environment,contributes to the defective osteogenesis of BMSCs and bone formation inestrogen deficiency-induced osteoporosis.The contents of the present study are as follows:Objective(1) Search for the key miRNA that regulate mBMSCs osteogenesis in themicroenviroment of estrogen-deficiency-induced osteoporosis (miR-21).(2) To investigate miR-21whether involved in the osteogenic differentiation ofmBMSCs and the effect in this progress in estrogen-deficiency-inducedosteoporosis.(3) To study the influence of inflammatory cytokines to miR-21and the mechanism of miR-21related with mBMSCs osteogenesis in themicroenviroment of estrogen-deficiency-induced osteoporosis.Methods(1) An ovariectomized animal model was employed. The osteogenicdifferentiation potential of mBMSCs derived from normal mice and OVXmice was investigated by Alizarin Red S and ALP staining. And osteogenicrelative genes determined by real time RT-PCR and western blot. E2andinflammatory cytokines levels in serum were examined via Elisa.(2) Screen the key miRNA (miR-21) through combination of bioinformaticsMethods and MicroRNA gene chip technology in the mBMSCs derived fromOVX and Sham mice. Real time RT-PCR was Used to detect the levels ofmiR21in these types of mBMSCs. The miR-21function was investigated bytransfecting pre-miR-21and anti-miR-21into mBMSCs. And osteogenicgene and protein expression was determined by Alizarin Red S, Oil red Ostaining, real time RT-PCR and Western blot analysis, respectively.(3) mBMSCs were treated by IL-1β (1ng/mL), IFN-γ (0.1ng/mL) and TNF-α(10ng/mL) during osteogenic differentiation. Real time RT-PCR was used todetect the levels of miR-21. Compared to the microRNA gene chip results,the major cytokines (TNF-α) was selected. After upregulation the expressionof miR-21in the presence of TNF-α, the osteogenic potential of mBMSCswere investigated.(4) E2and TNF-α neutralizing antibody were injected in OVX mice and thelevels of TNF-α and miR-21were measured. Micro-CT was used to examinethe bone in the femurs of the mice. Results(1) OVX model were sucessfully build. BMD of OVX group was lower thanSham group. The osteogenic potential of OVX-mBMSCs was poorer thanSham-mBMSCs. Levels of E2decreased and inflammatory cytokinsincreased in OVX mice.(2) miR-21were screened. Real time RT-PCR shows that miR-21inOVX-mBMSCs decreased compared with Sham-mBMSCs. UpregulationmiR-21in OVX-mBMSCs，RT-PCR, western blot, Alizarin Red S and ALPstaining suggested the potential osteogenesis of mBMSCs were enhanced.(3) TNF-α were the major inflammatory cytokine that effect miR-21.Upregulation miR-21in mBMSCs treated with TNF-α, RT-PCR, western blot,Alizarin Red S and ALP staining suggested miR-21can rescue the potentialof mBMSCs treated with TNF-α.(4) E2and TNF-α neutralizing antibody were injected in OVX mice and thelevels of TNF-α were reduces and miR-21were elevated. Micro-CT showedbone formation in the femurs of the mice treated with TNF-α neutralizingantibody were higher than control group.Conclusion(1) miR-21was the key miRNA that regulate mBMSCs osteogenesis in themicroenviroment of estrogen-deficiency-induced osteoporosis.(2) miR-21could promote the potential of mBMSCs in nomal and estrogendeficiency-induced osteoporosis.(3) In estrogen deficiency-induced osteoporosis, TNF-α was a key inflammatorycytokine that inhibited osteoblastic bone formation by suppressing miR-21expression and miR-21over-expression partially rescued the attenuated osteogenic ability of mBMSCs.(4) TNF-α-induced miR-21suppression may be one of mechanisms by whichTNF-α contributes to impaired bone formation In PMOP.