The Study On The Role And Mechanism Of Hypoxia-associated CircDEN ND2A/miR-625-5p/HMGA1 Regulatory Pathway In The Aggressiveness Of Glioma

Objective:As a newfound type of non-coding RNA,circular RNAs?circRNAs?are involved in various physiological and pathological processes via regulation of gene expression.Increasing evidence shows that aberrantly expressed circRNAs play a crucial role in the initiation and progression of many tumors.Hypoxia is a critical driver for glioma progression.However,the functions of different circRNAs in gliomas remain elusive.The purpose of this study is to investigate the expression of circRNA in glioma under hypoxia,and the role and mechanism of circRNA in promoting malignant progression of glioma.Methods:1.The levels of circRNAs,miRNAs,and mRNAs were quantified by qPCR.2.The intensity and localization of circRNA in glioma cells were analyzed by Fluorescence in situ hybridization.3.The proteins were detected by western blot.4.The binding sites of miRNA at circRNA and mRNA were studied by luciferase reporter assay.5.The direct interaction between circRNA and miRNA was determined by pull-down assay.6.The migratory capability of glioma cells were examined by wound healing assay.7.The invasive capability of glioma cells were examined by Transwell assay.8.Immunohistochemistry was performed to analyze protein level in glioma tissues.Results:We predicted circDENND2A?has_circ₀002142?to be a hypoxia-responsive circRNA in glioma via a bioinformatic analysis.We found that hypoxia induced the expression of circDENND2A,which promoted migration and invasion of glioma cells.To understand the behaviors of circDENND2A in glioma,we studied the putative miRNAs targeted by circDENND2A and examined the effect of circDENND2A on the expression of these miRNAs.The results showed a significant inhibition of miR-625-5p by circDENND2A overexpression,suggesting circDENND2A as an efficient sponge of miR-625-5p in glioma cells.Phenotype experiments verified that circDENND2A was required for the hypoxia-induced migration and invasion of glioma cells and that this occurred by sponging of miR-625-5p.To explore the downstream effectors targeted by miR-625-5p,we examined whether miR-625-5p regulates the expression of HMGA1 in glioma cells.As expected,miR-625-5p inhibits HMGA1 expression.Phenotype experiments showed that both overexpression of circDENND2A and knockdown of miR-625-5p enhanced the migratory and invasive capabilities of glioma cells,while silence of HMGA1 disrupted the malignant transformation.Clinical analysis indicated that the glioma tissues overexpressing HIFla exhibited a high expression of circDENND2A and HMGA1 as well as a low expression of miR-625-5p.In addition,circDENND2A was positively correlated with HMGA1,and miR-625-5p was negatively correlated with circDENND2A and HMGA1.Conclusions:Hypoxia-induced circDENND2A sponged miR-625-5p to increase the expression of HMGA1,thereby enhancing the malignant behaviors of glioma cells.