| Objectives:To investigate the effect of METTL3(methyltransferase like 3)on the biological characteristics and angiogenesis of endothelial progenitor cells(EPCs),and to explore whether METTL3 could influence the angiogenesis of EPCs through the methylation of m6 A,which provides a reference for further exploring the regulatory mechanism of vascular differentiation of EPCs.Methods:1.Tissue immunofluorescence staining was used to detect the expression of methyltransferase METTL3 at the Distraction Osteogenesis group and Bone Fracture group.2.The isolation,culture and functional identification of EPCs from dog bone marrow in vitro: after obtaining EPCs from young dogs by low density centrifugation:(1)The morphological characteristics of EPCs were observed under inverted microscope;(2)The intracellular markers of EPCs were identified by DIL-FITC double fluorescent staining;(3)The tube forming ability of EPCs was tested by Matrigel;(4)Transwell detected the migration function of EPCs;3.Immunofluorescence staining was used to detect whether METTL3 was expressed and localization in EPCs.4.After construction of lentivirus METTL3 overexpression and knockdown vector and transfected into dog EPCs,the optimal MOI was determined;the cell cycle of EPCs was detected by cell cycle flow cytometry;the transfection efficiency of overexpression and knockdown of METTL3 was detected by qRT-PCR and Western blot.5.CCK8 was used to detect the proliferation ability of EPCs in normal control group(Control group),overexpression negative virus control group(OE-Control group),overexpression group(OE-METTL3 group),knockdown negative virus control group(sh-Control group)and knockdown group(sh-METTL3 group);Matrigel tube forming experiment and Transwell were used to detect the effect of METTL3 on the tube forming ability and migration function of EPCs;The methylation level of m6 A in each group were detected by Colorimetric to explore the correlation between the expression of METTL3 and m6 A methylation modification.In addition,qRT-PCR was used to detect the expression of miR-21,miR-27 b,miR-205,miR-486 and the mRNA and protein expression of VEGF,bFGF and PI3K/AKT signaling pathway,to study the relationship between the expression of METTL3 and mature miRNA and its effect on the expression of VEGF,bFGF and PI3K/AKT signal pathway.Results:1.The expression of METTL3 in new born tissue of Distraction Osteogenesis group was higher than Bone Fracture group.2.Isolation and culture in vitro and identification of EPCs according to biological characteristics: The morphology of EPCs identified as long fusiform or polygonal under microscope;DIL-FITC double fluorescence staining showed that EPCs had the ability to absorb acetylated low-density lipoprotein and bind ulex europaeus agglutinin-1;Matrigel tube forming experiment showed that EPCs laid on Matrigel had the ability to form tube-like structure;EPCs had a certain migration ability,and cell migration of EPCs increased with time.3.METTL3 was positive in EPCs and mainly located in the nucleus.The expression of METTL3 was positively correlated with the methylation abundance of m6 A.The higher content of METTL3 was,the higher the methylation degree of m6 A was.And,the methylation abundance of m6 A was related to the functional characteristics of EPCs.The increase of the expression of METTL3 increase the methylation abundance of m6 A,which promoted the proliferation,migration and angiogenesis of EPCs.The expression of METTL3 decreased caused the decrease of m6 A methylation abundance,which inhibited the proliferation,migration and angiogenesis of EPCs.4.The results of qRT-PCR of miRNA showed that the expression of miR-21 increased with the increase of METTL3(P<0.01),and decreased with the decrease of METTL3(P <0.05),the difference was statistically significant;the expression of miR-27 b increased with the increase of METTL3(P <0.05),and decreased with the decrease of METTL3(P<0.05),the difference was statistically significant;There was no significant difference in the expression of miR-205 and miR-486 between the OE-METTL3 group and sh-METTL3 group(P >0.05).5.Compared with the control group,the results of mRNA qRT-PCR and Western blot showed that the mRNA and protein expression level of VEGF,bFGF,PI3 K and AKT were in direct proportion to the level of METTL3,and were up-regulated in OE-METTL3 group,while down-regulated in sh-METTL3 group.And the difference was statistically significant.Conclusions:1.The results showed that METTL3 was related to the methylation abundance of m6 A,and regulated the expression of miR-21 and mir-27 b by methylation modification of m6A;2.METTL3 could activate PI3K/AKT signaling pathway through m6 A methylation,thus promoting the angiogenesis of EPCs... |
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