The Impact And Mechanism Of Myoblast Exosomes Mediated MiRNA On The Osteoblastic Differentiation

Muscles and bones often change synchronously in osteoporosis,myosculosis and other diseases,and there is a close relationship.There is a positive feedback loop mechanism between adjacent muscles and bones,which regulates each other by secreting regulatory factors,and there exists intercellular signal transduction at cellular level,but all belongs to secreted proteins till now.Exosome as a vesicle secreted by cells,can mediate a variety of active molecules into receptor cells to play a regulatory role.Exosome play a role in the enrichment of miRNA,and the structure of exosome membrane can protect miRNA from RNA enzyme degradation,functional miRNA has multiple target regulatory effects.It has been shown that exosome can mediate functional miRNA into receptor cells to play a regulatory role.However,there is no report on the study of the interaction between skeletal muscle and bone in the exosome angles.Objective : This study aimed to determine the effect of myoblast exosome on osteogenic differentiation of osteoblasts,further exploration is made on the mechanism of mediated miRNA to promote bone differentiation,it provides new ideas and theoretical basis for the research of bone tissue engineering and the treatment of related diseases.Methods:(1)The exosomes of myoblast C2C12 cells was extracted and identified by transmission electron microscope and Western blot method from two aspects of morphology and molecular biology.(2)In order to detect the effect of C2C12 derived exosomes on the mineralization ability of MC3T3-E1 cells,the tracer experiment determined that the exosome was entered into the cells,add extracted exosomes get cultured MC3T3-E1 cells,and the osteogenesis model was established,alizarin red staining,ALP activity detection,Real-time fluorescence quantitative PCR was used to detect the osteogenic marker gene and ensure the model establishment.(3)High-throughput sequencing screened the main regulation of miRNA,transient transfection and downregulation of miRNA expression in C2C12 cells,Real-time fluorescence quantitative PCR to verify the transient transfection effect.(4)In order to verify the effect of myoblast derived exosome miR-27a-3p on the mineralization ability of MC3T3-E1 cells,extracted exosome from the transient transfection myoblast cells,and established the osteogenesis model again,and to detect the different osteogenic differentiation indexes,such as Alizarin red staining and ALP activity detection,to verified its effect on osteogenesis induction model.(5)In order to verify the effect of the change of miR-27a-3p expression in the exosome on the ?-catenin signaling pathway of receptor cells,Western blot detect the expression of APC and ?-catenin protein,and the nuclear transfer of ?-catenin protein was detected by Immunofluorescence.Results:(1)Transmission electron microscopy(TEM)revealed that the diameter of exosome was between 30-100nm;Western blot showed that the exosome expressed CD9,CD63 and CD81 labelled proteins.(2)The tracer results showed that the exosome into the cells and enriched around the nucleus;alizarin red staining results showed that a number of obvious mineralized nodules were produced after added the exosome of the induced culture,ALP activity test results showed that ALP activity increased significantly after added the exosome of the induced culture,which is time-dependent;the results of Real-time fluorescence quantitative PCR showed that the expression of ALP,OCN and Runx2 was elevated compared with the control group.(3)High-throughput sequencing results screened out the regulatory role of miRNA or miR-27a-3p;Real-time fluorescence quantitative PCR results showed that compared with the control group,the miR-27a-3p content of C2C12 cells and exosome was reduced significantly after 72 hours of instant transfection;extraction of exosomes after transfection,and added to recipient cells after cultured for 48 hours,the expression of miR-27a-3p was no obvious difference with the control group.(4)Compared with the control group,there was no significant difference in alizarin red staining,ALP activity and real-time fluorescence quantitative PCR between C2C12 cells transfected with inhibitor derived exosomes,which was significantly different from that of C2C12 derived exosomes.(5)The results of Western blot and Immunofluorescence showed that C2C12 cells derived exosome group the expression of APC was decreased,?-catenin protein expression was up-regulated,nuclear transfer increased;compared with C2C12 cells transfected with inhibitor derived exosome group and control group,there was no significant difference in the expression of APC and ?-catenin protein.Conclusions: The exosome of myoblast was successfully extracted,and the experimental results showed that the exosome of myoblast could promote osteogenic differentiation of osteoblasts.By changing the exosome content of miR-27a-3p,established the osteogenic differentiation model,and the osteogenesis phenotype was used to confirm that the miR-27 a by exosome mediated into osteoblasts,promotes the bone differentiation,and it was proved that myoblast exosome mediated miR-27a-3p through the regulates Wnt/?-catenin signaling pathway plays a role in osteogenic differentiation.It was first time explains the defection of muscle on bone from the perspective of exosomes,and this effect is realized by miR-27a-3p direct regulation of APC/?-catenin signaling pathway.