| Objective: In recent decades,the morbidity,disability and mortality of diabetes mellitus(DM)have risen sharply.Because of various harmful stimuli,diabetic patients eventually suffer from diabetic vascular complications,such as retinopathy,renal dysfunction,foot ischemic ulcer and gangrene.Among them,Ischemic foot ulcer and gangrene are mainly caused by severe hypoperfusion,which is related to vascular ischemic injury.Therefore,promoting angiogenesis and increasing blood perfusion is a therapeutic strategy for diabetic foot.MicroRNAs(miRNAs)are endogenous non-coding small RNAs that regulate gene expression after transcription and play an important role in cell proliferation,tube formation and migration.Studies have shown that miRNA-328 can regulate endothelial-mesenchymal transition under high glucose condition,participate in the process of atrial remodeling after atrial fibrillation,and inhibit tumors.The research aim was to observe the angiogenesis of miRNA-328 in diabetic ischemic cell model and to explore its mechanism.Methods: We cultured umbilical vein endothelial cells under high glucose and low serum for 24 hours to simulate the vascular ischemia induced by diabetes mellitus.The proliferation ability,migration ability and tube formation of umbilical vein endothelial cells cultured under high glucose and low serum condition and normal condition for 24 hours were detected byCCK-8 cell proliferation activity test,scratch test and tube formation test,respectively.The expression of microRNA-328 in human umbilical vein endothelial cells cultured under high glucose low serum condition and normal condition was detected by RT-qPCR.The expression of vascular endothelial growth factor(VEGF),a marker of angiogenesis,was observed by Western Blot assay.After transfection of NC and miR-328 inhibitor into HUVECs to achieve the purpose of control and down-regulation of miR-328 expression level,and both groups were cultured in high glucose and low serum condition for 24 hours,the proliferation,migration and tube formation of umbilical vein endothelial cells under high glucose and low serum conditions were detected by CCK-8 cell proliferation assay,scratch assay and tube formation assay,at the same time,Western Blot experiment was used to observe the expression of VEGF and related pathway proteins in the process of angiogenesis after down-regulation of miR-328;AKT pathway inhibitor Periforsin inhibits AKT phosphorylation and then observes the changes of endothelial cell tube formation and AKT/mTOR pathway.According to the experimental results of endothelial cell function,the target genes of miR-328 were predicted in the miRWalk database.The target genes intersection of Target Scan,miRanda,miRDB,miRWalk as a predictor of target genes,then Gene Ontology function enrichment analysis and KEGG Pathway analysis were performed in the DAVID database(https://david.ncifcrf.gov/).Finally,through reading the literature,the target genes of microRNA-328 regulating the HUVECs angiogenesis under high glucose and low serum conditions were screened out,finally,the target gene was validated by WB experiment.Results: Endothelial cell angiogenesis function test showed that: In the CCK-8 experiment,the proliferation activity of HUVECs in high glucose and low serum group was lower than that in the normal control group,it is 0.65 times as much as that in the normal control group(p=0.0004);In the cell wound scratch assay,the migration ability of HUVECs in high glucose and low serum group was lower than that in the normal control group,it is 0.81 times as much as that in the normal control group(p=0.0003);In tube formation test,the microtubule formation activity of HUVECs in high glucose and low serum group was lower than that in the normal control group,it is 0.45 times as much as that in the normal control group(p <0.0001);RT-qPCR showed that the expression of microRNA-328 in HUVECs cultured in high glucose and low serum was 4.2times higher than that in normal control group(p <0.0001).The result of Western blot showed that the VEGF expression level of HUVECs cultured in high glucose and low serum was lower than that of normal control group,which was 0.4223 times than that in normal control group(p <0.0001).After transfection of NC and miR-328 inhibitor,and both groups were cultured in high glucose and low serum condition for 24 hours: In the CCK-8 experiment,the proliferation activity of HUVECs in the miR-328 inhibitor group was higher than that in the NC group,it is 1.19 times as much as that in the NC group(p =0.0069);In the cell wound scratch assay,the migration ability of HUVECs in the miR-328 inhibitor group was higher than that in the NC group,it is 1.23 times as much as that in the normal control group(p =0.0038);In tubeformation test,the microtubule formation activity of HUVECs in the miR-328 inhibitor group was higher than that in the NC group,it is 1.28 times as much as that in the NC group(p =0.0006);The result of Western blot showed that the VEGF expression level of HUVECs in the miR-328 inhibitor group was higher than that in the NC group,it is 3.337 times as much as that in the NC group(p=0.0001).Western Blot assay showed that the ratio of p-AKT/AKT protein in high glucose and low serum group was 0.76 times than that in control group(p< 0.0001).It was concluded that the AKT signal pathway of HUVECs was inhibited under high glucose and low serum conditions.After transfection of NC and miR-328 inhibitor,the ratio of p-AKT/AKT protein in the miR-328 inhibitor group was 4.1 times higher than that in the NC group(p < 0.0001).When AKT phosphorylation was inhibited by Periforsin,endothelial cell tube formation was inhibited and AKT/mTOR signaling pathway was inhibited(P <0.05).In the predicted results of target genes,183 target genes of miR-328 were preliminarily identified in the miRWalk database.Further analysis of Gene Ontology and KEGG Pathway showed that the target genes of miR-328 were mainly related to cell response to glucose,cell proliferation,cell migration,angiogenesis,cell cycle arrest,and so on.By consulting the literature,the target genes that may be associated with endothelial cell angiogenesis are PIM1,MMP16,LYVE1,PTEN.We selected PIM1 for validation,WB experiment showed that down-regulation of microRNA-328 could promote PIM1 protein expression in endothelial cells(P < 0.05).KEGG Pathway analysis shows that there are eight cellular pathways regulated by microRNA-328,includingPI3K-Akt signaling pathway?Insulin resistance?AMPK signaling pathway and so on,in which the AKT pathway has been verified to be relevant to this research.Conclusion: 1.High glucose and low serum conditions inhibit angiogenesis of endothelial cells(inhibiting proliferation,migration and angiogenesis of HUVECs),and promote the expression of microRNA-328.2.Downregulation of microRNA-328 in high glucose and low serum conditions can promote angiogenesis of endothelial cells(promoting proliferation,migration and angiogenesis of HUVECs).3.The mechanism of microRNA-328 regulating angiogenesis of endothelial cells under high glucose and low serum conditions is the target gene PIM1 and AKT/mTOR signaling pathway. |
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