| Background: The incidence rate of fracture nonunion,failure of spine fusion,osteoporosis were getting higher and higher with the increasing of the aging society,the rapid development of the transportation industry.These conditions have serious impact on the quality of life of the patients,which has been the major problem for orthopedic surgeon.Detailed understanding of the regulative molecular pathways of bone formation,which Is a complex physiological regulation process,not only can decrease the occurrence of osteoporosis,promote the healing of bone fractures,but also can shorten the spinal fusion time and achieves a better fusion rate and early clinical results.Osteoblasts cell as the main functional cells of bone formation,is responsible for the synthesis,secretion and mineralization of bone matrix.The timing and sequence of differentiation of mesenchyma stem cell are tightly controlled,bone formation and this regulation model is realized by the signal pathway which is very complex.Through thorough,comprehensive study and understanding of the molecular signaling pathway of osteoblast differentiation is an important foundation for bone tissue engineering and is conducive to further study of osteogenic differentiation.The canonical Wnt signaling pathway controls normal embryonic development,cellular growth and proliferation,which plays an important role in the regulation of osteoblast development that are derived from mesenchymal stem cells with multiple differentiation potential.Wnt is a secreted glycoprotein,Wnt signaling pathway could increase the accumulation of intracellular beta chain protein(beta-catenin).Beta-Catenin as a multifunctional protein,involved in the formation of the adhesive tape.Free beta –catenin plays an important part in regulation of gene expression by the action of enter the nucleus.The glycoprotein secreted by Wnt signaling pathway plays a critical role in the development of many tissues,especially in bone tissue.Micro RNA is a class of endogenous non-coding single stranded small RNA molecules containing 22 nucleotides,which could be combined with the mRNA 3 ’non translation region of target gene.O n the one hand,RNA interference silencing RNA complex(RISC)can be formed by binding to intracellular proteins,which specifically bind and degrade the target mRN A by sequence.O n the other hand,the translation can be inhibited by non complete complementary binding,which can regulate gene expression at the post transcriptional level.There are about one thousand of miRN A sequences,up to now,which are involved in many physiological and pathological processes,such as the development of organisms,cell differentiation,proliferation,apoptosis,tumor occurrence,inflammation and so on.In addition,it is speculated that one third of gene may be regulated by the expression of miRNA.More and more evidences indicate that miRNA gene expression at the level of post translational modification plays an important role in the regulation of the differentiation of MSCs into osteoblasts.There are some miRNAs,for example,miR-96,miR-124,miR-199 a,miR-100,miR-138,miR-204 and miR-20 a,are involved in the study of osteogenic differentiation.MiR-101 is usually quiescent in human tumor cells,whereas a large number of studies show that miR-101 plays a fundamental role in the metabolism of many cells,especially in the regulation of cell proliferation and differentiation.However,the molecular mechanism of osteoblasts differentiation of MSCs by miR-101 remains unclear.Recently,new evidences indicate that EZH2 inhibits Wnt/β-catenin signaling pathway inactivation by inhibiting the Wnt gene,thus blocking the differentiation of MSCs into osteoblasts.Objective:Through the primary culture of bone marrow mesenchymal stem cells in vitro and the induction of differentiation of osteoblasts,dynamic detection of miR-101 changes in the process of cell proliferation and cell differentia tion.Using inhibitors and analog of miR-101,the effects of changing miR-101 levels(high expression and low expression)on the osteogenic differentiation of MSCs were observed,and also,the changes of EZH2 related target genes were observed.Moreover,t he changes of differentiation of hBMSCs induced miR-101 and the effect on the activity of Wnt/ beta-Catenin signal pathway were also observed,through the changes of EZH2 expression level.It is foundational to reveal the relationship between miR-101,EZH2 and Wnt/β –Catenin,and provide a strong evidence for miR-101 in the regulation of mesenchymal stem cells differentiating into osteoblasts.Methods : 1.MiR-101 in the osteogenic differentiation of adult bone marrow mesenchymal stem cells.Using adult bone marrow mesenchymal stem cells were purchased from C yagen biotechnology company between the original generation method to observe the role of miR-101 in steogenic differentiation.The expression level of miR-101 in osteogenic differentiation of hBMSCs was observed by q RT-PCR technique.The specific markers of bone cells(such as: RUNX2,ALP,OPN and OCN)expression,ALP activity,cell mineralization level etc were detected,through the expression changes of bone marrow mesenchymal stem cells by the lentivira l transfection technique,which could reflect the role of miR-101 in the osteogenic differentiation of hBMSCs.2.MiR-101 of EZH2 adjustment.We first analyzed the miR-101 and EZH2’s 3 ’UTR region by bioinformatics prediction,and found that the over expression of miR-101 can significantly reduce the protein expression of EZH2 by blot Western.EZH2 may be a direct target gene of miR-101,and miR-101 can inhibit the expression of EZH2.3.EZH2 regulating miR-101 mediated osteogenic differentiation of adult bo ne marrow mesenchymal stem cells.We interference the expression level of EZH2 in adult bone marrow mesenchymal stem cells by plasmid construction,then,the activity of ALP and expression of osteoblast specific markers were detected by q RT-PCR analysis technology.4.Wnt signaling pathway in miR-101 to adjust the role of osteogenic differentiation in adult bone marrow mesenchymal stem cells.The activity of Wnt signaling pathway was detected by TOPflash/FOPflash technique,and the changes of Wnt signal pat hway after intervention of miR-101 and EZH2 were observed.Results:1.Expression of miR-101 is increased during osteogenic differentiation of hBMSCs,2.miR-101 promotes osteoblast differentiation of hBMSCs,3.miR-101 directly targets EZH2 and inhibits its expression,4.EZH2 mediate miR-101-regulated osteogenic differentiation of hBMSCs,5.miR-101 promotes osteogenic differentiation of hBMSCs by targeting EZH2 and regulating Wnt/β-Catenin pathway.Conclusions: Overall,our data demonstrated that miR-101 promoted osteogenic differentiation of hBMSCs by targeting EZH2,which resulted in activation of Wnt/β-Catenin signaling pathway.Our findings provide a new insight that miR-101 possesses the great potential as a novel class of therapeutic target for bone regeneration. |
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