Effects And Mechanisms Of MiRNA-411-5p On The Migration And Tube Formation Of Glioma-exposed Microvascular Endothelial Cells

ObjectiveGlioma is the most common primary malignant tumor in the central nervous system.Although great progress has been made in surgery,chemotherapy and radiotherapy,the prognosis of patients with malignant glioma is still unsatisfactory.Glioma is a typical vascular dependent solid tumor,its growth mainly depends on tumor angiogenesis,and angiogenesis is the main form of glioma angiogenesis.Therefore,how to inhibit the angiogenesis of glioma is a hot topic in molecular targeted therapy of glioma,which has been widely concerned by scholars at home and abroad.MicroRNA(miRNA)is a kind of noncoding endogenous RNA with a length of 22 ~ 24 nucleotides.It plays a regulatory role by targeting the 3’untranslated regions(3’UTR)of its downstream target gene to degrade mRNA or inhibit mRNA translation.The study showed that many differentially expressed miRNAs were involved in the angiogenesis of gliomas.MiR-411 is a member of the 14q32.31 miRNA gene cluster.Many members of the gene cluster play an important role in neovascularization.However,it is not clear whether miR-411 is involved in the regulation of glioma angiogenesis.There have been studies that miR-411 was significantly down-regulated in glioma tissues and cells,and it was involved in the regulation of glioma cell proliferation by evolutionarily conserved non coding RNA,suggesting that miR-411 might be a potential tumor suppressor gene.However,the expression of miR-411 in glioma-exposed microvascular endothelial cells(GECs)is not clear.Whether miR-411 can inhibit glioma angiogenesis by inhibiting the migration and tube formation of glioma-exposed microvascular endothelial cells,has not been reported.Angiogenic factor with G patch and FHA domains 1(AGGF1)is a newly found angiogenic factor in the study of Klippel Trenaunay syndrome(KTS).AGGF1 is highly expressed in vascular endothelial cells and is involved in endothelial cell proliferation,migration,tube formation and aortic ring centered angiogenesis.Our previous studies showed that AGGF1 is involved in the regulation of glioma angiogenesis.However,it is not clear whether AGGF1 is involved in the effect of miR-411-5p on the regulation of glioma angiogenesis.In this study,the expression level of miR-411-5p in glioma-exposed microvascular endothelial cells was firstly determined.Secondly,the effects of miR-411-5p on the migration and tube formation of glioma-exposed microvascular endothelial cells was further investigated.Finally,whether miR-411-5p could affect the angiogenesis of glioma by targeting AGGF1 was investigated,which provided a new target for anti-angiogenesis therapy of glioma.MethodsThe glioma-exposed microvascular endothelial cells were prepared with U87 glioma conditioned medium.The expression of miR-411-5p in glioma-exposed microvascular endothelial cells was detected by real-time quantitative polymerase chain reaction(qRT-PCR).Overexpression or silencing of miR-411-5p were performed by transfecting miR-411-5p agomir or antigomir respectively.QRT-PCR was used to verify the transfection efficiency.Scratch test,Transwell cell migration test and Matrigel tube formation test were used to detect the migration ability and tube formation ability of glioma-exposed microvascular endothelial cells,and to further study the effect of miR-411-5p on the angiogenesis of glioma.The wild-type and mutant-type AGGF1 3’UTR reporter gene plasmids were constructed and co-transfected with miR-411-5p agomir into 293 T cells respectively.The target binding of miR-411-5p and AGGF1 was analyzed by double luciferase reporter gene system.QRT-PCR and western blot were used to detect the mRNA and protein expression of AGGF1 after overexpression or silencing of miR-411-5p.ResultsThe expression of miR-411-5p in glioma-exposed microvascular endothelial cells was lower than that in normal microvascular endothelial cells(P<0.05 vs.ECs group).The expression level of miR-411-5p was significantly up-regulated in the cells transfected with miR-411-5p agomir(P<0.01 vs.miR-411-5p agomir NC group);the expression level of miR-411-5p was not significantly down-regulated in the cells transfected with miR-411-5p antiagormir(P>0.05 vs.miR-411-5p antagomir NC group).Overexpression of miR-411-5p significantly inhibited the migration(P<0.05 或P<0.01 vs.miR-411-5p agomir NC group)and tube formation ability(P<0.01 vs.miR-411-5p agomir NC group)of glioma-exposed microvascular endothelial cells,while silencing of miR-411-5p significantly promoted the migration(P<0.05 或P<0.01 vs.miR-411-5p antagomir NC group)and tube formation(P<0.01 和 P<0.05 vs.miR-411-5p antagomir NC group)ability of glioma-exposed microvascular endothelial cells.MiR-411-5p can target AGGF1(P<0.05 vs.AGGF1-wt + miR-411-5p NC group).Overexpression of miR-411-5p significantly decreased the mRNA(P<0.05 vs.miR-411-5p agomir NC group)and protein(P<0.01 vs.miR-411-5p agomir NC group)expressions of AGGF1,while silencing of miR-411-5p significantly increased the mRNA(P<0.01 vs.miR-411-5p antagomir NC group)and protein(P<0.01 vs.miR-411-5p antagomir NC group)expressions of AGGF1.Conclusions1.The expression of miR-411-5p in GECs was significantly down-regulated.2.Overexpression of miR-411-5p significantly inhibited the migration and tube formation ability of GECs,and silencing of miR-411-5p significantly promoted the migration and tube formation ability of GECs.3.MiR-411-5p can target AGGF1 and down-regulate its expression level,which maybe the mechanism of miR-411-5p inhibiting the angiogenesis of glioma.