Evaluation Of MIR-29C Inhibits Endotheliocyte Migration And Angiogenesis Of Human Endothelial Cells By Suppressing The Insulin Like Growth Factor 1

MicroRNAs, a class of 22-nucleotide non-coding RNAs, modulate gene expression by associating with the 3’-untranslated regions (3’-UTRs) of messenger RNAs (mRNAs). Although multiple miRNAs are known to be regulated during angiogenesis, their individual roles in blood vessel development are still not fully understood. Herein, we investigate the role of miR-29c in regulating cell cycle and angiogenic phenotype of endothelial cells. The results showed that IGF-1 is highly expressed and down-regulated by miR-29c in human umbilical vein endothelial cells (HUVEC). Consistent with this preliminary finding, introduction of exogenous miR-29c or miR-29c inhibitor alters cell cycle progression, proliferation and tube formation of HUVEC, respectively. Furthermore, by using luciferase reporter assay, we find that the expression of IGF-1, a suppressor transcription factor, is directly regulated by miR-29c through 3’-UTR. As a conclusion, we find that miR-29c plays a significant role in regulating cell cycle, proliferation and angiogenic properties of HUVECs. This function is likely mediated through IGF-1 proteins at the post-transcriptional level. As a novel molecular target, miR-29c may have a potential value in the treatment of angiogenesis-associated diseases, such as cardiovascular diseases and cancers.Part I Affection of miR-29c in expression of vascular formation related transcription factorObjective:To investigate the affection of miR-29c in expression of vascular formation related transcription factor.Methods:Human umbilical vein endothelial cells (HUVECs) were cultured and transfected with miR-29C mimics and miR-29C inhibitor. mRNA of relevant factor expression after miRNA transfection were detected by Real-time PCR while associated factor protein expression were detected by Western Blot after miRNA transfection, and expression of VEGF were detected by immunofluorescence.Results:Human umbilical vein endothelial cells transfected with miR-29c mimics caused PI3K, AKT and significantly decreasion of VEGF mRNA and protein expression levels (P<0.01), but after IGF-1 added there only occurred protein levels dcreasion, without mRNA expression significant change(P> 0.05). Immunofluorescent cell staining results showed that human umbilical vein endothelial cells transfected with miR-29c mimics could inhibit VEGF expression(P<0.05), whereas transfection of miR-29c inhibitor increased expression of VEGF (P<0.05).Conclusions:miR-29c can inhibit mRNA and protein expression of angiogenesis-related transcription factor PI3K, AKT and VEGF. Meanwhile, miR-29c can reduce IGF-1 protein expression, but does not affect its mRNA expression, suggesting that the role of reducing IGF-1 is a post-transcriptional regulation, which means translation inhibition.Part II Affection of miR-29c in human umbilical vein endothelial cell proliferation, migration and angiogenesisObjective:To investigate the affection of miR-29c in human umbilical vein endothelial cell proliferation, migration and angiogenesis.Methods:Human umbilical vein endothelial cells (HUVECs) were cultured and transfected. MTT assay and EDU were used to detect cell proliferation, flow cytometry were used to detect cell cycle. Transwell assay were used to detect cell migration, and tubule formation assay were used to detect angiogenic capabilities.Results:In human umbilical vein endothelial cells transfected with miR-29c mimics group, HUVECs and 293-T proliferation rate of the cells declined 57.6%and 36.3%respectively, there was a significant difference (P<0.05) with the control group control; group transfected with miR-29c mimetic compared with the rest of the group, the number of DNA synthesis in S phase of the cell were lowest, while the G2 phase of the cell number were the most; HUVECs proliferation activity decreased; HUVECs migration capability significantly reduced compared with the control group decreased by 31%, compared with transfer transfected miR-29c inhibitor group decreased 64%; cell angiogenesis significantly decreased while angiogenic vessels are smaller in diameter and loop rate decreased (P <0.05). Group transfected with miR-29c inhibitor compared with the remaining groups, had the largest number of celsl of in DNA synthesis S phase; HUVECs proliferation activity was significantly enhanced; HUVECs migrating ability was markedly increased; cell angiogenesis have strengthened (P<0.05).Conclusions:miR-29c can inhibit human umbilical vein endothelial cell proliferation, migration and angiogenesis, which may be due to the miR-29c-expression affection in vascular endothelial growth factor signaling pathways. Then the biological behavior of human umbilical vein endothelial cells was affected.Part Ⅲ Study about the molecular mechanisms of miR-29c inhibits the angiogenesis of human umbilical vein endothelial cellsObjective:To Study about the molecular mechanisms of miR-29c inhibits the angiogenesis of human umbilical vein endothelial cells.Methods:miRNA target genes were predicted biologically after culturing of human umbilical vein endothelial cells (HUVECs). Molecular mechanisms of miR-29c inhibiting human umbilical vein endothelial cell angiogenesis were study by luciferase reporter gene assay which built IGF-1 3’UTR luciferase plasmid and cell scratch experiment.Results:miR-29c mimics significantly decreased fluorescence intensity of IGF-1 3’UTR luciferase reporter gene (P<0.05); but it could not reduce the fluorescence intensity of IGF-1 3’UTR mutation group’s luciferase reporter gene (P> 0.05). After adding miR-29c inhibitor, fluorescence intensity of IGF-1 3’UTR luciferase reporter gene significantly increased (P<0.05), but the mutation group did not change significantly compared with the control group (P> 0.05). Cell scratched 24h, miRNA-29c mimics transfected human umbilical vein endothelial cells (HUVECs) group had a significantly slower healing speed than that of the miRNA-29c inhibitor transfected group (P<0.01); when human umbilical vein endothelial cells (HUVECs) was transfected with miRNA-29c mimics and cultured with IGF-1, their cratch healing speed increased significantly (P<0.05). IGF-1 prompted to save the miRNA-29c human umbilical vein endothelial cells’(HUVECs) reduced migration ability.Conclusions:IGF-1/PI3K/AKT/VEGF pathway can regulate angiogenesis, miRNA-29C targeted bind in IGF-1 3’UTR end, thereby inhibiting the expression of IGF-1 in the translational level. The inhibition of IGF-1 reduced downstream AKT pathway-related genes expression such as PI3K, and VEGF, and ultimately inhibit the capacity of blood vessel formation of human umbilical vein endothelial cells (HUVECs).