The Mechanism Of The Tumor Growth And Invasion Regulated By Circ-U2AF1 Through Hsa-miR-7-5p/NOVA2 Signaling Pathway In Human Glioma

Objective:We aimed to assess the relationship between circ-U2AF 1 and hsa-miR-7-5p and examine hsa-miR-7-5p-mediated NOVA2 effects on proliferation,apoptosis and the metastatic of glioma cells regulated by neuro-oncological ventral antigen 2(NOVA2),and investigate its biological activity and mechanism in vitro and in vivo.To determine the correlation the circ-U2AF 1/hsa-miR-7-5p/NOVA2 regulatory pathway may be a novel therapeutic target for treating gliomas and provide basis for clinical application.Methods:The expression levels of circ-U2AF1,hsa-miR-7-5p and NOVA2 in human glioma tissues and glioma cell lines were evaluated by qRT-PCR.Circ-U2AF1 siRNA was transfected into glioma cell lines U87MG and U251 to silence circ-U2AF1.The expression efficiency of circ-U2AF1 was detected by PCR.The proliferation,invasion and apoptosis of U87MG and U251 cells were detected by MTT assay,migration and invasion assay,cell cycle and apoptosis analysis.At the same time,the expression changes of hsa-miR-7-5p and NOVA2 in glioma cell lines were detected after the silencing of circ-U2AF1.The effect of silencing circ-U2AF1 on the expression of NOVA2 in glioma cell lines was analyzed by Western blot.The interaction between circ-U2AF1 and hsa-miR-7-5p and and the possible targeting relationship between NOVA2 and hsa-miR-7-5p was verified by double luciferase gene reporting system.Whether circ-U2AF1 has specific enrichment effect on hsa-miR-7-5p was further verified by biotin pull-down assay.To verify the mechanism of the circ-U2AF1/hsa-miR-7-5p/NOVA2 regulatory pathway affecting the biological behavior of glioma cells,subsequently the effects of on proliferation,invasion and apoptosis of glioma cells were analyzed by silencing hsa-miR-7-5p.the NOVA2,which is the possible target gene of hsa-miR-7-5p,was further silenced to evaluate the effects of on proliferation,invasion and apoptosis of glioma cells,in the same time,silencing NOVA2 and over-expression of hsa-miR-7-5p had the same effect.These experiments in vitro demonstrated that hsa-miR-7-5p and NOVA2 affect the biological behavior of glioma cells in circ-U2AFl/hsa-miR-7-5p/NOVA2 pathway.Finally,we established a subcutaneous glioma model in nude mice to investigate its anti-tumor effect in vivo,and detected the effect of apoptotic effects of p53,Bax and Bcl-2 on the growth of glioma by Western blot,in other to verified the effects of over-expression of circ-U2AF1 and silencing of hsa-miR-7-5p on NOVA2 regulation effect of glioma growth.Results:The results showed that circ-U2AFl and NOVA2 were markedly upregulated in glioma tissues of different grades compared to normal brain tissues,Additionally,we observed that circ-U2AF1 and NOVA2 in glioma cell lines U87MG and U251 were higher than the normal glial cell line HEB.On the contrary hsa-miR-7-5p expression was significantly decreased in glioma tissues and glioma cell lines U87MG and U251 compared to normal brain tissues and HEB cells,respectively.The knockdown efficiency in both cell lines transfected with circ-U2AFl siRNA displayed that transfection with the circ-U2AF1 siRNA significantly decreased the expression of circ-U2AF1 in glioma cells.MTT assay revealed that silencing of circ-U2AF1 decreased the proliferation of U87MG and U251 cells compared to the control vector group.Through flow cytometry analysis,we observed that circ-U2AFl knockdown upgraded the apoptosis of both glioma cell lines relatively to the control vector group.Cell cycle analysis indicated that silencing of circ-U2AF1 markedly increased the number of cells in G1 phase and decreased that of cells in G2 phase compared to cells transfected with the control siRNA,suggesting that silencing of circ-U2AF1 blocks cell cycle at the G0/G1 phase to subsequently induce cell apoptosis.Silencing circ-U2AF1 significantly changed the expression levels of hsa-miR-7-5p and NOVA2 mRNA,with hsa-miR-7-5p being unregulated and NOVA2 down regulated.Furthermore,western blotting analysis showed that NOVA2 expression was decreased in both cell lines transfected with circ-U2AF1 siRNA.Luciferase reporter assays showed that cells co-transfected with the wild-type circ-U2AF1 reporter and the hsa-miR-7-5p mimic showed a significant decrease in relative luciferase activity compared to cells co-transfected with the hsa-miR-7-5p negative control.However,cells co-transfected with the mutant circ-U2AF1 reporter and hsa-miR-7-5p mimic did not exert any effect on reporter luciferase activity.To further verify whether circ-U2AF1 sponges miR-7-5p,the biotin pull-down assay was performed.The results showed that higher levels of circ-U2AFlbound to miR-7-5p could be detected in the Bio-miR-7-transfected cells by qRT-PCR compared to the control cells,suggesting that circ-U2AF1 can bind to and pull down miR-7.In addition,luciferase reporter assays showed that cells co-transfected with the wild-type NOVA2 3’-UTR reporter and the hsa-miR-7-5p inhibitor showed a significant decrease in relative luciferase activity compared to cells co-transfected with the hsa-miR-7-5p negative control.However,cells co-transfected with the mutant NOVA2 reporter and hsa-miR-7-5p mimic did not exert any effect on reporter luciferase activity.Compared to the control inhibitor group,the hsa-miR-7-5p inhibitor led to increased cell proliferation,migration and invasion,and inhibited the apoptosis of glioma cells while blocking the cell cycle at G0/G1 phase.Silencing of NOVA2 and over-expression of hsa-miR-7-5p inhibited the proliferation and induced apoptosis of U251 cells,Moreover,NOVA2 silencing and over-expression of hsa-miR-7-5p significantly inhibited the migration and invasion of glioma cells.We found that tumor volumes and weights in the circ-U2AF1 vector group and hsa-miR-7-5p inhibitor group were significantly higher compared to the control groups in vivo.Western blotting analyses of the resulting tumors showed that NOVA2 expression level was increased in the circ-U2AF1 vector and hsa-miR-7-5p inhibitor groups.In addition,we found that p53 and Bax expression levels were decreased while the expression level of the anti-apoptotic protein Bcl-2 was increased in the circ-U2AF1 vector and hsa-miR-7-5p inhibitor groups in vivo.Conclusion:Circ-U2FA1 exerted oncogenic role in glioma cells.Furthermore,Circ-U2AF 1 promotes human glioma via derepressing hsa-miR-7-5p-mediated neuro-oncological ventral antigen 2 up-regulation.We suggested that circ-U2AF1 could be a prognostic biomarker and the circ-U2AF1/hsa-miR-7-5p/NOVA2 regulatory pathway may be a novel therapeutic target for treating glioma.