| Objective: Distraction osteogenesis(DO)technology is to place distractors on both sides of the broken end of bone to achieve the regeneration of blood vessels,tissues and new bone through slow traction tension,so as to prolong and repair bone tissue.Mi RNAs is made up of 21-23 nucleotide single endogenous non-coding RNA molecules,they with target genes mRNA 3 ’-the translation section(3’ UTR)of base pairing complementary sites),negative regulation of gene expression is well known,the blood vessels of the newborn is the premise and foundation for the formation of new bone,and miRNAs in regulating the structure and function of embryonic and postnatal vascular development plays an important role,the miRNA-486 was recently identified as miRNAs associated with red tie.Therefore,in this study,I will explore the influence of miRNA-486 on the biological characteristics of EPCs in vitro,verify the target gene of miRNA-486 to explain whether miRNA-486 affects the angiogenic differentiation of EPCs by targeting TIE1,and the regulatory mechanism of miRNA-486 on the activation of the angiogenic signaling pathway of EPCs.It provides new therapeutic targets and theoretical basis for the treatment of distraction osteogenesis.Methods: 1.Isolation,culture and identification of EPCs and the influence of miRNA-486 on EPCs angiogenesis: Density gradient centrifugation was used to isolate cultured EPCs,and the cells were identified by DIL-FICT fluorescent double staining.Matrigel tubular formation assay,Transwell migration assay,flow cell cycle assay were used to measure the tubular formation capacity,migration and proliferation of EPCs.Lentiviral vectors with overexpression of miRNA-486(LV-Cfa-Mir-486)and lentiviral vectors with inhibition of miRNA-486(LV-CFA-Mir-486-inhibition)were constructed to transfected EPCs respectively to determine the optimal conditions for virus infection.The silencing and overexpression efficiency of lentivirus transfected EPCs were measured by RT-qPCR.The proliferation,migration and tubule-forming abilities of EPCs in the blank Control group(Control,Control)and the experimental group(LV-Mir-486 or LV-Mir-486-inhibitor)were measured by flow cell cycle assay(Transwell)and Matrigel tubular experiment.RT-qPCR and Western blot were used to detect the relative expression levels of mRNA and protein of VEGF and bFGF2.Identification of miRNA-486 target gene TIE1 and the research of miRNA-486 on activation of PI3K/Akt pathway: The target gene TIE1 was verified by double luciferase reporter assay.The relative expression of TIE1 in each group was tested by RT-qPCR and Western blot,respectively,to further verify the effect of miRNA-486 on target gene TIE1.Western blot was used to detect the influence of miRNA-486 on the expression of key factors of the PI3 K and Akt,key proteins of the downstream PI3K/Akt signaling pathway.Results:1.The adherent wall of isolated and cultured cells showed colony growth,and the cell morphology showed spindle,polygon or spindle shape.The fusiform EPCs induced in vitro were interlinked to form several hollow tubular networks.EPCs have the ability to engulf DIL-AC-LDL and combine FITC-UEA-1 to make the cells double stained with red-green coincidence fluorescence.In the cell migration experiment,the number of cells passing through the Transwell compartment microporous membrane increased with time.Flow cell cycle assay showed that EPCs had the potential of proliferation and differentiation and had the characteristics of stem cells.When MOI was 50,the infection efficiency of lentivirus transfected cells reached 80% after 72 hours and the cell growth was good.RT-qPCR detected mir-486 silencing and overexpression after lentivirus transfection with EPCs with high efficiency,which could meet the requirements of subsequent experiments.In the migration experiment after transfection of EPCs,the number of EPCs overexpressing miRNA-486 through the Transwell chamber microporous membrane was relatively high,while the number of miRNA-486 silenced EPCs migrated to the Transwell chamber was relatively low.In flow cell cycle experiments after transfection of EPCs,overexpression of miRNA-486 can promote DNA synthesis and cell division of EPCs,while silencing expression of miRNA-486 can inhibit cell growth.Matrigel tubule formation experiment found that there were more tubule structures of EPCs overexpressing miRNA-486 and fewer lumen structures of silenced miRNA-486 EPCs.RT-qPCR and WB detection of the expression of vascular related genes after transfection of EPCs showed that overexpression of miRNA-486 would up-regulate the mRNA and protein levels of bFGF and VEGF,and silencing miRNA-486 would reduce the mRNA and protein levels of bFGF and VEGF.2.Dual luciferase reporter gene experiment results: miRNA-486 can specifically bind to the target site in the 3’UTR region of TIE1 mRNA and inhibit the production of reporter gene protein.RT-qPCR and Western blot results of TIE1 after lentivirus transfection of EPCs: the relative mRNA expression level and protein expression level of TIE1 in EPCs that overexpressed miRNA-486 were significantly lower than those in the negative control group,while the transcription level and protein translation level of TIE1 in EPCs that down-regulated miRNA-486 were notably higher than those in the negative control group.Relative protein expression of key factors of the PI3K/Akt pathway after EPCs transfection was detected by Western blot.The protein levels of PI3 K and Akt increased after the up-regulation of miRNA-486expression;conversely,protein levels of PI3 K and Akt were inhibited in EPCs down-regulated by miRNA-486 expression.Conclusion:1.Mi RNA-486 promoted the proliferation,migration and tubule-forming capacity of EPCs,as well as the expression of VEGF and bFGF of vascule-related genes.Mi RNA-486 was involved in the vascule-forming differentiation of EPCs.2.MiRNA-486 can target and regulate the TIE1 gene and participate in the activation of the PI3K/Akt signaling pathway,thereby promoting the proliferation and angiogenesis differentiation of EPCs in vitro. |
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