MicroRNA-576-3p Inhibits The Migration And Proangiogenic Abilities Of Hypoxia-treated Glioma Cells Through Hypoxia-inducible Factor-1?

Objectives:The present study aimed to investigate the effects of miR-576-3p on the migration and proangiogenic abilities of hypoxia-treated glioma cells.By in vitro observation of the number of cells in the lower layer of the Transwell chamber,the relative number of migrating cells in the scratch test,and the state of tube formation in the angiogenesis experiment,to analyze the effects of miR-576-3p on glioma migration and angiogenesis in hypoxia.In order to analyze the relevant mechanism of action,and provide new insights for clinically relevant treatment of glioma,we analyzed the targeting relationship between miR-576-3p and HIF-1? through the double luciferin report.Methods:Using real-time fluorescent quantitative polymerase chain reaction(RT-PCR)to detect miR-576-3p expression in clinical glioma samples and hypoxic-treated glioma cells,and analyze whether there is a difference in miR-576-3p expression in different glioma levels and hypoxic culture conditions.By constructing a lentivirus-stably transfected glioma cell line to knock down the expression of miR-576-3p and explore its effect on the hypoxia-treated glioma cell migration and angiogenic ability.In addition,miRWalk,miRanda and TargetScan website online tools are used to predict miR-576-3p candidate targets.The interaction between miR-576-3p and HIF-1? was verified by dual luciferase reporter gene,Western Blot and RT-PCR.Lentivirus-stably transfected cell lines were constructed to knock down the expression of HIF-1?,and its effects on the hypoxia-treated glioma cell migration and angiogenic ability were studied.Western Blot and ELISA kits were used to detect the expression of MMP2 and VEGF.Finally,the effect of exogenous overexpression of HIF-1? on the hypoxiatreated glioma cell migration and angiogenesis-promoting ability based on miR-576-3p overexpression was investigated using a recovery experiment.Results:(1)Compared with glioma specimens,miR-576-3p was significantly increased in non-cancerous brain tissue,and miR-576-3p was significantly down-regulated in hypoxia-induced glioma cell lines.(2)The results of the scratch test,Transwell and angiogenesis experiments showed that compared with the control group,the miR-576-3p lentivirus overexpressing cell line significantly reduced the migration and angiogenesis of glioma cells under hypoxic conditions.(3)The online tools of miRWalk,miRanda,and TargetScan predict that miR-576-3p and HIF-1? interact with each other,and dual luciferase reporter gene analysis and western blot analysis indicate that exogenous overexpression of miR-576-3p can significantly reduce the protein expression of HIF-1? in glioma cell lines under hypoxic conditions by direct targeting of HIF-1? 3'-UTR.(4)The results of the scratch test,Transwell and angiogenesis experiments show that compared with the control group and the blank group,the knockdown of HIF-1? expression under hypoxic conditions can significantly inhibit the migration and angiogenic ability of glioma cells.(5)Recovery experiments showed that the recovery of HIF-1? expression attenuated the effects of miR-576-3p on the migration and angiogenesis of glioma cells,and the corresponding protein expression levels of MMP2 and VEGF also changed.Conclusion:This study confirmed that miR-576-3p is a new type of glioma inhibitor that can inhibit the malignant progression of glioma under hypoxic conditions.By targeting the expression of HIF-1?,it can effectively inhibit the hypoxia-induced glioma cell migration and the ability to promote angiogenesis.