Circ＿TRPS1 Promotes Malignant Progression Of Glioma By Sponging MiR-149-3p And Up-Regulating VEGFA Expression

Objective:In order to further clarify the mechanism of malignant progression of glioma and explore new therapeutic targets,this study was carried out and focused on the expression profile of circ RNAs in glioblastoma,expression level of circ＿TRPS1 in glioblastoma,the role of circ＿TRPS1 in regulating the malignant biological behavior of glioma and the molecular mechanism.Materials and Methods:Transcriptome sequencing was performed to identify the circ RNAs differently expressed between 3 glioblastoma tissues and 3 normal brain tissues collected during posterior cranial decompression.Candidate targeted circ RNAs were selected according to false discovery rate(FDR)and fold change(FC).Then,gene ontology analysis was utilized to identify the biological processes,sellular components,and molecular functions potentially associated with circ RNAs abnormally expressed in glioblastoma while KEGG(Kyoto encyclopedia of genes and genomes)pathway analysis was carried out to detect the pathway enrichment.Quantitative reverse transcription polymerase chain reaction(q RT-PCR)was utilized to detect the expression levels of the candidate targeted circ RNAs(including circ＿TRPS1,circ＿ASAP1,circ＿QKI and circ＿PTK2)in 3 glioblastoma multiforme tissues and 3 normal brain tissues.Then,we detect the expression level of the targeted circ RNA(circ＿TRPS1)in 40 glioma tissues(including diffuse astrocytoma,anaplastic astrocytoma and glioblastoma multiforme),6 paracancerous tissue and 6normal brain tissues by q RT-PCR.Data of clinical and pathological features were collected.The association between circ＿TRPS1 expression level and clinical as well as pathological features was statistically analyzed.Sanger sequencing was performed following polymerase chain reaction(PCR)to detect the back splicing point of circ＿TRPS1.q RT-PCR was used to confirm the resistance of circ＿TRPS1 to RNase R digestion.After we designed the divergent primers and convergent primers of circ＿TRPS1 complementary DNA(c DNA)and genomic DNA(g DNA),PCR and agarose gel electrophoresis were carried out to confirm the closed loop structure of circ＿TRPS1.Because the efficiency of circ＿TRPS1 interference was not satisfying,we identified the effect of circ＿TRPS1 on malignant progression of glioma by circ＿TRPS1 overexpression.A lentiviral vector with overexpression of circ＿TRPS1was constructed and applied to establish stable circ＿TRPS1 overexpression U87 glioblastoma cell line.The expression level of circ＿TRPS1 was detected by q RT-PCR following circ＿TRPS1 overexpression.The effect of circ＿TRPS1 on the proliferation of U87 glioma cells was evaluated by CCK8 proliferation assay.Transwell migration assay was carried out to determine the impact of circ＿TRPS1on glioma cell migration.Western blot(WB)and q RT-PCR was performed to identify the impact of circ＿TRPS1 on epithelial-mesenchymal transition(EMT)of glioma cells by evaluating the expression levels of E-cadherin and Vimentin.Then,nude mouse glioma subcutaneous transplantation models were established.Tumor size and body weight were collected to confirm the effect of circ＿TRPS1 on tumor growth in vivo.The targeted mi RNA and the downstream m RNA were predicted by bioinformatics analysis.q RT-PCR and immunofluorescence were utilized to determine the expression levels of the targeted mi RNA(mi R-149-3p)and the downstream m RNA(vascular endothelial growth factor A,VEGFA)in glioma tissues,paracancerous tissue and normal brain tissues.Association between expression of mi R-149-3p as well as VEGFA and pathological grade were statistically analyzed.The effect of circ＿TRPS1 overexpression on the expression level of mi R-149-3p and VEGFA was determined by q RT-PCR and WB.Dual luciferase reporter assay was carried out to confirm the interaction between circ＿TRPS1 and mi R-149-3p and the interaction between mi R-149-3p and VEGFA.Following VEGFA overexpression,CCK8 proliferation assay and transwell migration assay were performed to determine the impact of VEGFA on proliferation and migration of glioma cells,respectively.Rescue experiments were carried out to confirm the effect of circ＿TRPS1-mi R-149-3p-VEGFA axis on malignant progression of glioma.Results:A total of 4069 and 9661 circ RNAs were detected in glioblastoma tissues and normal brain tissues by transcriptome sequencing,respectively.There were 710 circ RNAs abnormally expressed in glioblastoma,of which 256 circ RNAs were overexpressed.Gene ontology analysis predicted that biological processes associated with circ RNAs abnormally expressed in glioma included phosphorylation,protein transportation,chromatin modification,etc.;molecular function included protein binding,ATP binding,nucleotide binding,etc.;and cellular components included cytoplasm,nucleoplasm,nucleus,etc.Pathway analysis found that the circ RNAs abnormally expressed in glioma were related to phosphatidylinositol signaling system,RNA degradation,m RNA surveillance pathway,erythroblastic leukemia viral oncogene(Erb)B signaling pathway,etc.circ＿TRPS1,circ＿ASAP1,circ＿QKI and circ＿PTK2 were identified as candidate targeted circ RNAs according to FDR and FC.q RT-PCR revealed that the expression level of circ＿TRPS1 was significantly higher in glioma tissues compared with paracancerous tissue and normal brain tissues while the expression levels of circ＿ASAP1,circ＿QKI and circ＿PTK2 were slightly lower in glioma tissues.The expression level of circ＿TRPS1 was higher in high grade glioma compared with low grade glioma.However,circ＿TRPS1 expression levels in anaplastic astrocytoma and glioblastoma multiforme were similar.Kaplan-Meier survival analysis suggested that patients with glioma in higher circ＿TRPS1 expression group had significantly shorter OS than those in lower circ＿TRPS1 expression group.Sanger sequencing confirmed the back splicing point of circ＿TRPS1.The resistance of circ＿TRPS1 to RNase R digestion was confirmed by q RT-PCR.Agarose gel electrophoresis following PCR revealed that the c DNA of circ＿TRPS1could be amplified by both convergent primer and divergent primer,while the g DNA of circ＿TRPS1 could be amplified by the convergent primer,but could not be amplified by the divergent primer,which confirmed the closed loop structure of circ＿TRPS1.We constructed a circ＿TRPS1 overexpressed lentiviral vector to establish stable U87 glioblastoma cells overexpressing circ＿TRPS1,which was confirmed by q RT-PCR.CCK8 proliferation assay showed that circ＿TRPS1 overexpression promoted cell proliferation in glioma.Transwell migration assay found that circ＿TRPS1 overexpression promoted cell migration in glioma.q RT-PCR and WB found that compared with the control group,the expression level of E-cadherin were significantly lower while the expression level of Vimentin was significantly higher in circ＿TRPS1 overexpression group,which indicated that circ＿TRPS1overexpression promoted the EMT of glioma.The in vivo experiments found that the tumor size in glioma subcutaneous transplantation model was larger in circ＿TRPS1 overexpression group when compared with that of the control group.Bioinformatics analysis predicted that circ＿TRPS1 sponged mi R-149-3p while mi R-149-3p regulated the expression level of VEGFA m RNA.q RT-PCR found that the expression level of mi R-149-3p was lower in glioma tissues compared with paracancerous tissue and normal brain tissues and that the expression of mi R-149-3p was associated with pathological grade of tumor.Immunofluorescence and q RT-PCR revealed that VEGFA was overexpressed in glioma tissues and that higher VEGFA expression was associated with higher pathological grade of glioma.Moreover,mi R-149-3p expression was inversely correlated with circ＿TRPS1 expression while VEGFA m RNA expression was inversely correlated with mi R-149-3p expression in glioma tissues.Overexpression of circ＿TRPS1 down-regulated mi R-149-3p expression but up-regulated VEGFA expression.Dual luciferase reporter assay confirmed the interaction between circ＿TRPS1 and mi R-149-3p and the interaction between mi R-149-3p and VEGFA m RNA.CCK8 proliferation assay revealed that VEGFA overexpression promoted cell proliferation in glioma.Transwell migration assay showed that VEGFA overexpression promoted cell migration in glioma.In the rescue experiments,mi R-149-3p mimics remarkably attenuated the effect of circ＿TRPS1 overexpression on cell proliferation and migration in U87 glioma cell lines.The inhibition of cell proliferation and migration caused by mi R-149-3p mimics could be rescued by VEGFA overexpression.Conclusion:Expression profile of circ RNAs in glioblastoma was different from that in normal brain tissues.Abnormally expressed circ RNAs in glioblastoma may be involved in chromatin modification,nucleotide binding,phosphatidylinositol signaling system,RNA degradation,m RNA surveillance pathway,Erb B signaling pathway,etc.circ＿TRPS1 was overexpressed in glioma.Higher circ＿TRPS1 expression level was associated with higher pathological grade of glioma and shorter OS of patients.The expression level of mi R-149-3p was decreased in glioma tissues.The expression of mi R-149-3p was associated with pathological grade of glioma.VEGFA was overexpressed in glioma.Higher expression of VEGFA was associated with higher pathological grade of glioma.The back splicing point and the closed loop structure of circ＿TRPS1 were confirmed.circ＿TRPS1 promoted malignant progression of glioma by sponging mi R-149-3p and up-regulating VEGFA expression.These findings indicated that the circ＿TRPS1-mi R-149-3p-VEGFA axis might be a therapeutic target against malignant glioma.