Preliminary Study Of MicroRNAs Regulating Wnt Signaling Pathway Of Endothelial Progenitor Cells In Vasculogenesis During Distraction Osteogenesis

Objective: Distraction Osteogenesis(DO)has an incomparable osteogenic rate of fracture healing,but the osteogenic mechanism is not clear.In our previous study on distraction osteogenesis,we found that CD133,a surface marker of endothelial progenitor cells(EPCs),has high expression in the new bone region.Therefore,we hypothesized that DO might initiate the embryonic vascular repair model and participate in the new bone formation.Moreover,through high-throughput sequencing method also preliminary findings noncoding RNA in stage DO with embryonic angiogenesis has significant correlation.It was found that miRNA-21,miRNA-10,mi RNA-451,miRNA-27 b,miRNA-486 and miRNA-205 have common expression in the distraction osteogenesis group and the embryonic bone group,and there are significant differences with other groups(normal bone tissue,fracture tissue,etc.).Therefore,we speculate those microRNAs may regulate target genes of EPCs in vasculogenesis during Distraction osteogenesis.This study was based on the previous studies of distraction osteogenesis and screen out the miRNAs of EPCs during vascularization.Methods: 1.The research on in vitro isolation,cultivation,identification and vascular capacity of EPCs: Health hybrid dogs,3week,injected 1ml of 5% pentobarbital into the abdominal cavity for anesthesia,and then punctured in the bilateral tibia and pumped tibia bone marrow 5ml.Finally using density gradient centrifugation to obtain EPCs:(1)Observe the morphological characteristics of EPCs after primary and induced cultures by inverted phase contrast microscope;(2)Identification of surface markers by flow cytometry to identify EPCs;(3)Cell cycle flow cytometry was used to detect the proliferation of EPCs;(4)Transwell migration assay and cell scratch assay were used to detect the migration ability of EPCs.2.Screening of miRNAs regulating the vascularization of EPCs and its bioinformatics analysis: Select EPCs for vascularize 1,3,7,10,14,21,and 28 days.The pre-induction cell was served as a control group of “0” day.RNA was extracted from these cells and reversely transcribed using a miRNAs extraction kit.The qRT-PCR was performed to detect the relative expression of miRNA-10,miRNA-451,miRNA-486,miRNA-21,mi RNA-27 b and miRNA-205 during vasculogenesis in EPCs,and analysis of the dynamic expression of VEGF-A and bFGF by the same method.After observe these genes expression trends,we used statistical methods to analyze the correlation of miRNAs,VEGF-A and bFGF,in order to screen out the miRNAs of EPCs during vascularization.Finally,we continue to use bioinformatics to predict the target genes and signaling pathways which are regulating the vasculogenesis of EPCs.3.The differential expression of Wnt signaling pathway after EPCs receive mechanical and vasculogenic induction:EPCs were completely randomized into 4 groups as follows:A.blank group: static culture;B.mechanical force group: centrifugal force was loaded for 24 min(100 r/min),four times per day;C.vasculogenic induced group: induce with vasculogenic fluid;D.mechanical force and vasculogenic induced group: unite with group B and C.After 3 days,Performed qRT-PCR and WB to detect the expression of Wnt signaling pathway in EPCs.Results: 1.The research on in vitro isolation,cultivation,identification and vascular capacity of EPCs:(1)Morphology of EPCs: EPCs were able to grow adhering to the wall.A spindle-shaped pseudopodium was gradually formed over time.A large number of cells were arranged in a radioactive manner.Finally,the colonies are clustered into strips and eventually become ring-like structures;(2)Surface markers CD133 can be detected in EPCs;(3)Transwell migration and cell scratch experiments demonstrated that EPCs have a certain ability of migration.2.Screening of mi RNAs regulating the vascularization of EPCs and its bioinformatics analysis: They have statistically significant positive correlations between miRNA-486,miRNA-205,miRNA-21,mi RNA-27 b and VEGF-A,bFGF in the process of the vasculogenesis(p<0.05).Bioinformatic websites were used to perform target gene prediction and gene ontology(GO)and KEGG signaling pathway was used to analysis these miRNAs which relate vasculogenesis.(1)A total of 6,191 target genes of miRNA27 b were predicted by bioinformatic website,and 169 target genes were related to blood vessels.AGGF1,EPAS1,and BMPR2 are the common target genes among three bioinformatic websites;(2)The bioinformatic website predicted a total of 5003 target genes for miRNA-21,including 98 target genes associated with blood vessels;BMPR2 and JAG1 are the common target genes among three bioinformatic websites;(3)The bioinformatic website predicted a total of 7367 target genes for miRNA-205,including 169 target genes associated with blood vessels;FOXF1 and QKI are the common target genes among three bioinformatic websites;(4)The bioinformatic website predicted a total of 6040 target genes for miRNA-486,including 118 target genes associated with blood vessels;TIE1 is the common target gene among three bioinformatic websites.In the above results,we have found BMPR2 shared by microRNA-21 and micro RNA-27 b.The KEGG signaling pathway analysis of miRNA-27 b,miRNA-21,miRNA-205,and miRNA-486 revealed that the WNT signaling pathway and the FoxO signaling pathway are shared by the aforementioned microRNAs.3.The differential expression of Wnt signaling pathway after EPCs receive mechanical and vasculogenic induction: LRP5,AXIN,β-catenin and LEF1 in Wnt signaling pathway widely expressed after EPCs were induced by angiogenesis and mechanical stimulation.However,the mRNA expression of GSK-3β was contrary to the expression of other genes.The result of Western Blot showed that the protein of β-catenin and VEGF-A express accordingly with genes.Conclusion: 1.EPCs have the potential for forming blood vessels and we hypothesized that DO might initiate the embryonic vascular repair model and participate in the new bone formation;2.The miRNAs of mi RNA-486,miRNA-205,miRNA-21 and mi RNA-27 b may play an importent role in regulating the vascularization of EPCs during distraction osteogenesis;3.The miRNAs of mi RNA-486,miRNA-205,miRNA-21 and mi RNA-27 b may regulate Wnt signaling pathway and promote vasculogenesis by inhibiting the target gene.