The Role Of MiR-223-3p Regulates Human Bone Mesenchymal Stem Cells Osteogenic Differentiation

Objective:Osteoporosis(OP)is a type of systemic metabolic bone disease characterized by high morbidity,high harm,and prone to brittle fractures.One of the causes of osteoporosis is the weakened osteogenic differentiation ability and the enhanced adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMMSCs).Changing the direction of differentiation of bone marrow mesenchymal stem cells is a potential treatment for osteoporosis.MicroRNA is a kind of short sequence non-coding RNA that widely exists in eukaryotic cell organisms and regulates cell proliferation,differentiation and apoptosis by degrading the mRNA of target gene or inhibiting the translation.Recent studies have shown that miRNA plays an important regulatory role in the osteogenic differentiation of mesenchymal stem cells,and its role in osteogenesis has increasingly become a research focus.The previous research group found that miR-223-3p was up-regulated in mesenchymal stem cells of osteoporosis mice,but whether it caused the occurrence and development of osteoporosis has not been reported.This project aims to study the role of miR-223-3p in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMMSCs),predict and verify its target genes,and elucidate the molecular mechanism of miR-223-3p regulating osteogenic differentiation of human bone marrow mesenchymal stem cells,so as to provide a new theoretical basis for the diagnosis and treatment of osteoporosis.Methods:1.Separation and culture of primary human bone marrow mesenchymal stem cells:bone marrow was collected during total hip arthroplasty,then human bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow adherent method,their morphological characteristics were observed under the microscope.2.Verify the high-throughput sequencing results:the collected cells were divided into the osteoporosis group and the normal bone mass group,and the expression differences of miR-223-3p between the two groups were detected by qRT-PCR.3.Verify the role of miR-223-3p in osteogenic differentiation of mesenchymal stem cells:total RNA was extracted from stem cells at different time points of osteogenic differentiation,and the expression trend of miR-223-3p was detected by qRT-PCR.The endogenous expression of miR-223-3p in stem cells was changed by cell transfection and divided into four group:(1)blank control group.(2)NC control group;3.miR-223-3p mimics group;(4)miR-223-3p inhibitor groups.After osteogenic differentiation for 7 days,the expression of the osteogenic landmark genes Runx2 and OPN were detected.After osteogenic induction for 21 days,alizarin red staining was used to observe the expression of calcified nodules.4.Verify the miR-223-3p target genes:bioinformatics analysis and target gene prediction software were used to analyze downstream target genes that may bind to miR-223-3p,and target genes closely related to osteogenesis were screened in combination with literature.After inhibiting or overexpressing miR-223-3p,the protein expression of target genes were analyzed by Western Blot.Results:1.The primary human bone marrow mesenchymal stem cells isolated and cultured by the whole bone marrow adherent method grews slowly,and the cells were in the shape of long spindle or polygon.With the increase of the fluid changes,the cells were gradually purified.2.In mesenchymal stem cells of osteoporosis patients,the expression of miR-223-3p was significantly up-regulated.3.During osteogenic differentiation,with the extension of induction time,the expression of miR-223-3p gradually decreased.The expression of miR-223-3p mimics constitutive bone-related genes Runx2 and OPN were down-regulated,and the positive nodules of alizarin red staining were decreased.However,the expression of bone related genes of miR-223-3p inhibitor Runx2 and OPN were up-regulated,and the number of alizarin red staining nodules increased.4.The target gene prediction software found that FOXO1,which is closely related to osteogenic differentiation,is one of the downstream target genes of miR-223-3p,and further proved that FOXO1 is the target gene of miR-223-3p.Conclusions:1.The human bone marrow mesenchymal stem cells collected by whole bone marrow adherent separation method have good activity and high cell purity,which can be used as a method to obtain human bone marrow mesenchymal stem cells.2.miR-223-3p is up-regulated in osteoporosis stem cells and down-regulated in osteogenic differentiation.3.miR-223-3p inhibits osteogenic differentiation of mesenchymal stem cells by targeting the expression of FOXO1,thereby leading to the occurrence and development of osteoporosis.4.miR-223-3p may become a new clinical marker for the diagnosis and prognosis of osteoporosis,as well as a potential target for the treatment of osteoporosis.