The Mechanisms Of MicroRNA-24 In Influencing The Formation Of Human Umbilical Vein Endothelial Cell Lumen Through Autophagy

Objective1 To investigate the regulation of autophagy by micro RNA-24(mi R-24)and related molecular mechanisms.2 To explore the effect of miR-24 on the proliferation,migration and luminal formation of human umbilical endothelial cells(HUVECs)induced by rapamycin on the basis of autophagy.Methods1 Grouping of experiments,transfection of miR-24 high expression plasmid and establishment of autophagy modelAccording to the experimental requirements,HUVECs were randomly divided into control group,miR-24 high expression + rapamycin group,and rapamycin group.HUVECs in control group was not given any treatment.HUVECs in mi R-24 high expression + rapamycin group were first transfected with miR-24 high expression plasmid,and then used 1000 nmol/L rapamycin to intervene for 6h to establish the autophagy model.HUVECs in the rapamycin group were treated with 1000 nmol/L rapamycin for 6h to establish autophagy model.2 To detect the expression level of autophagy-related genes Beclin-1 and LC3 II and the production of autophagosomes in each experimental groupThe mRNA level of Beclin-1 and LC3? were detected by RT-PCR.Western blotting and immunocytochemistry were used to detect the protein level of Beclin-1 and LC3?.Transmission electron microscopy was used to observe the formation of autophagosomes in the cells.3 To detect the morphology,proliferation,migration and lumen formation of HUVECs in each experimental groupHE staining was used to observe the morphological changes of cells in each group.CCK-8 method was used to detect the proliferation of cells in each group.Scratch test was used to detect the migration ability of cells in each group.Then Matrigel assay was used to test the ability of lumen formation of cells in each group.Results1 Compared with the control group,the mRNA and protein expression levels of Beclin-1 and LC3 II in the rapamycin group were significantly increased(P<0.05),and more autophagosomes were observed in the cytoplasm.However,the mRNA and protein expression levels of Beclin-1 and LC3 II in miR-24 high expression + rapamycin group were only increased to a low extent(P<0.05),and there was only a small amount of autophagosomes in the cytoplasm.It can be seen that miR-24 can significantly inhibit the mRNA and protein expression of Beclin-1 and LC3 II,and inhibit the formation of autophagosomes.2 Compared with the control group,the morphology of the cells in the rapamycin group changed slightly,showing an irregular state with a few cells aging.The activity of cells,the number of migration,and the number of lumen-like structures were all decreased to a lesser extent(P<0.05).On the other hand,the cells in miR-24 high expression + rapamycin group had severe aging,and the activity,number of migration,and number of lumen-like structures were all significantly decreased(P<0.05).Conclusions1 miR-24 can directly inhibits the expression of autophagy-related genes Beclin-1 and LC3? at the transcriptional level,and it can also inhibits the formation of autophagosomes,then downregulate the level of autophagy.2 miR-24 can significantly inhibit the proliferation,migration and lumen formation of HUVECs which induced by rapamycin,the mechanism may be related to the existence of autophagy.Down-regulation of autophagy may be one of the mechanisms by which miR-24 inhibits the lumen formation of cell.