MiR-705Regulates The Abnormal Differentiation Of Bone Marrow Mesenchymal Stem Cells Derived From Osteoporotic Postmenopausal Mice

Caused by estrogen deficiency, postmenopausal osteoporosis (OPM) is a generalmetabolic disease due to imbalance between osteoclast-related bone resorption andosteoblast-related bone formation which has led to bone loss and reform, and thereforehigh fracture risk. Domedtic aged women bears an50%OPM incidence. Osteoporosis isone of the major factors in the loss of the jaw bones. The subsequent complicationsinclude alveolar bone absorption, gingival atrophy, exposure of tooth root, loss ofattachment of abutment tooth, loosening of abutment tooth and atrophy of jaw bones,which bring difficulties in denture and implant rehabilitation. However, current therapieshave been failing to reach ideal efficacy because of side effects including poor absorption,instable effect, low long term patient compliance and persistence.As a sort of adult stem cells from mesoderm, bone marrow mesenchymal stemcells(BMMSCs) have acted critically in bone development, regeneration and repair.BMMSCs sever as the source of osteoblast and lipocyte, and the balance of osteo-andadipogenesis capacity thereof is closely correlated with bone remodeling homeostasis. Thedecline in proliferation and osteogenesis along with increase in adipogenesis of BMMSCsmight be the fundamental reason of OPM, but the exact mechanism remains unknown. Being prevalent in animal and plant cells, microRNA, a sort of uncoding small RNArecently found, bears the ability of silencing target genes by binding to mRNA3’-untranslated regions, through which way determining stem cells’ fate and exertingimportant influence on osteo-or adipogenesis. But, which cluster of miRNA have playedthe key role in BMMSCs differentiation, along with their signaling pathway and effect inOPM is still unsolved. Our study has conducted BMMSCs comparison from normal andOPM mice and microRNA microarray analysis to explore function of differentiallyexpressed miRNA, with which the role of miRNA in OPM and a deep understanding ofmechanism of OPM may be released to provide threads and methods for new strategies ofOPM treatment and improvement, then it can provide new insights into prevention andtreatment of loss of the jaw bones and can give instructions in the choose of restorationdesign.Objective:(1) To detect changes in osteo-and adipogenesis capacity of OPM BMMSCs.(2) To pick out miRNA with differential expression in O-BMMSCs miRNAtranscription profile.(3) To figure out the function of the picked out miRNA in O-BMMSCs osteo-andadipogenesis, as well as its target genes and mechanism involved.(4) To determine the restoration of O-BMMSCs osteo-and adipogenesis differentiationthrough restoring miRNA to normal level.Methods:(1) OPM mice model was successfully established by ovariectomy. BMMSCs wereisolated from bone marrow of both O-BMMSCs and S-BMMSCs to compare theirbiological characters.(2) miRNA microarray was employed to get both O-BMMSCs and S-BMMSCstranscription profile, whose results were later confirmed by qRT-PCR to pick outspecific miRNA.(3) miR-705was both over-expressed and inhibited using transfection to figure out itseffect on BMMSCs: using MTT to detect proliferation effect; red oil O, alizarin redand ALP stain, qRT-PCR, as well as Western blot were adopted respectively to examthe effect on osteo-and adipogenesis capacity of BMMSCs. Prospective target genesof miR-705were first picked out from bioinformatics websites and target-geneprediction softwares and later identified by Western blot, qRT-PCR and luciferase enzymes’ activity.(4) Transfection was used to down-regulated miR-705transcription in O-BMMSCs,so as to reversed their differentiation potency on osteo-and adipogenesis, which hadbeen later examed by oil red O, alizarin red and ALP stain, qRT-PCR along withWestern blot.Results:(1) Both O-BMMSCs and S-BMMSCs have normal mesenchymal stem cell surfacemarkers and no hematopoiesis cell markers. O-BMMSCs bear lower cloningefficiency compared with S-BMMSCs, with obvious lower reproductive activity asdemonstrated by population doubling time and mitotic cycle, ALP stain, alizarin redstain and osteo-related genes detection after osteogenesis induction suggestsO-BMMSCs a poorer osteogenesis capacity, while a stronger adipogenesis as shownby oil red O stain and adipo-related genes detection after adipogenesis induction(2) Ten microRNAs have differential expression in S-BMMSCs and O-BMMSCs,among which miR-705, miR-3077-5p and miR-20a hold the greatest disparity.miR-705，whose elevated expression in O-BMMSCs has conformed to RT-PCRresults, showed decreased expression in physical osteogenesis and increasedexpression in adiposeness and also enjoyed higher abundance in bone. We decided tochoose miR-705for research.(3) Experiments on function of miR-705didn’t suggest any effect on BMMSCs growthcycle from both its over-expression and inhibition. While up-regulation of miR-705expression could reduce osteogenesis capacity and enhance adipogenesis capacity ofBMMSCs, down-regulation could in turn, enhance osteogenesis capacity and reduceadipogenesis capacity. According to bioinformatics prediction, well-definedosteogenesis-promoting gene Hoxa10and Foxo1are probable target genes, whoseexpression change inversely with miR-705and can bind to miR-705with mRNA3’-untranslated regions.(4) Down-regulated expression of miR-705could result in enhanced osteogenesiscapacity and depressed adipogenesis through not only promoting expression ofFoxo1, Hoxa10and other critical osteogenesis genes, but also reducing expression ofcritical adipogenesis genes.Conclusions: (1) While BMMSCs from OPM mice retained fundamental characteristics of stemcells, it showed reduced osteogenesis and enhanced adipogenesis capacity.(2) There was obvious differences in miRNA expression between BMMSCs from OVXmice and sham-operated mice.(3) miRNA acted positively on adipogenesis and negatively on osteogenesis ofBMMSCs, whose underlied mechanism might be the negative regulation of miR-705on its target gene Hoxa10, Foxo1.(4) In OPM progress, miR-705’s over-expression breaks the balance of BMMSCsosteogenesis and adipogenesis, therefore reduces their bone formation capacity, it isalso the direct reason for reduction of bone mass and lower quality of jaw bones andskeleton.