The effect of circ-ZEB1 on the biological characteristics of glioma cells

Abstract

Objective To investigate the effects of circ-ZEB1 on proliferation, invasion and migration of glioma cells. Methods Real-time quantitative PCR (polymerase chain reaction) was used to detect the expression of circ-ZEB1 in 30 glioma tissues and 8 normal brain tissues(obtained from Department of Neurosurgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University), glioma cell lines (A172 and U251) and normal glial cells (HA1800). A172 and U251 cells were transfected with si-circ-ZEB1 to knock down circ-ZEB1. According to the type of si-circ-ZEB1, it was divided into si-1 group and si-2 group (transfected with si-circ-ZEB1-1, si-circ-ZEB1-2, respectively), and a control group was established (siRNA-NC as negative groups transfected with siRNA-NC). The effect of circ-ZEB1 on the proliferation of glioma cell lines was detected by CCK-8 assay. Scratch assay and Transwell assay were used to observe the effects of circ-ZEB1 on the migration and invasion in glioma cells respectively. CircNet database was used to examine and evaluate of circRNA-miRNA gene regulatory network of circ-ZEB1. The dual luciferase reporter assay was used to validate the interaction of circ-ZEB1/miR-140-3p in the gene regulatory network of circRNA-miRNA. Results The expression of circ-ZEB1 was higher in glioma tissues than in normal tissues (12.16±1.31 vs. 6.84±0.56, P=0.001). Circ-ZEB1 was over-expressed in high-grade glioma tissues compared with low-grade gliomas (13.77±3.27 vs. 7.50±1.82, P=0.001). The expression levels of circ-ZEB1 in A172 and U251 were 2.91±0.30 and 3.32±0.20, respectively, compared with HA1800 (both P<0.01). The results of CCK-8 showed that the growth of circ-ZEB1 knockdown group (si-1 group and si-2 group) was significantly inhibited compared with the control group. The proliferation abilities of A172 cells were decreased by 40.0% and 57.1%, respectively (both P<0.01) and those of U251 cells were decreased by 35.3% and 48.5%, respectively (both P<0.01). In the scratch test, the migration of circ-ZEB1 knockdown group was significantly inhibited (both P<0.05). Transwell assay showed that the invasive abilities of glioma cells in si-1 group and si-2 group were lower than those of the control group (both P<0.01). The gene regulatory networks of circRNA-miRNAs showed that circ-ZEB1 interacted with a variety of miRNAs, which affected the regulation of multiple miRNAs and their downstream proteins. The dual luciferase reporter assay also confirmed the interaction of circ-ZEB1/miR-140-3p in the gene regulatory network of circRNA-miRNA. Conclusions Circ-ZEB1 is over-expressed in glioma tissues and cell lines. Down-regulation of circ-ZEB1 could decrease cell proliferation and cell migration in gliomas. The gene regulatory network of circRNA-miRNA suggests that circ-ZEB1 functions by interacting with a variety of miRNAs. Key words: Glioma; Circ-ZEB1; Cell proliferation; Cell migration assays