A novel circular RNA circENTPD7 contributes to glioblastoma progression by targeting ROS1

Abstract

Background: Circular RNAs (circRNAs) are identified to play an important role in many human cancers, such as glioblastoma. However, the potential mechanisms underlying the relationship between circRNAs and glioblastoma pathogenesis are still elusive. This study is designed to investigate the role of circRNAs in glioblastoma progression.

Methods: The present study is designed to investigate the mechanism by which circRNAs involves in glioblastoma pathogenesis. By using circRNAs microarray, we detected the dysregulated circRNAs and identified an up-regulated circRNA, circENTPD7 in glioblastoma tissues. Cell proliferation was measured using a CCK-8 assay. Cell clone formation ability was assessed with a clone formation test. We used the bioinformatics website to predict circRNA-miRNA and miRNA-mRNA interactions. CircRNA-miRNA interaction was confirmed by dual-luciferase reporter assays and RNA-RNA pulldown assay.

Results: circENTPD7 (hsa_circ_0019421) was upregulated in glioblastoma tissues. Kaplan-Meier survival analysis indicated that glioblastoma patients had a poor overall survival when circENTPD7 expression levels were high. Knockdown of circENTPD7 inhibited the motility and proliferation of glioblastoma cells. Moreover, we demonstrated that circENTPD7 acted as a sponge of miR-101-3p to regulate the expression of ROS1 further promoted the proliferation and motility of glioblastoma cells.

Conclusions: Taken together, these findings indicate that circRNA circENTPD7 promotes glioblastoma cell proliferation and motility by regulating miR-101-3p/ROS1.

Keywords: Glioblastoma; ROS1; circENTPD7; circRNA; miR-101-3p.

Fig. 1

circENTPD7 is overexpressed in the glioblastoma tissue and cells. a The heatmap showed the representative dysregulated circRNAs in glioblastoma tissues analyzed by microarray. b CircENTPD7 (hsa_circ_0019421) was upregulated in the glioblastoma cells compared to the normal cells. c We could only amplify back-spliced forms of ENTPD7 from cDNA by PCR using divergent primers following by gel electrophoresis. The canonical linear forms of ENTPD7 in both and gDNA and were amplified in both cDNA and gDNA. d RT-qPCR for the abundance of circENTPD7 and ENTPD7 mRNA in glioblastoma cells treated with RNase R were performed, ENTPD7 mRNA rather than circENTPD7 were decreased after RNase R treatment. e CircENTPD7 was most localized in the cytoplasm by FISH analysis in U87 cells. f CircENTPD7 was enriched in U87 cytoplasm fraction. Levels of circENTPD7, ENTPD7 mRNA, GAPDH, and U6 RNA in purified U87 nuclear and cytoplasm fractions were detected by RT-qPCR. Data are presented as mean ± SD, Student’s t test, ***P < 0.001

Fig. 2

The upregulation of circENTPD7 in osteosarcoma predicts poor prognosis. a RT-qPCR analysis was carried out to detect the expression level of circENTPD7 in 90 glioblastoma tissues (n = 90) and paired noncancerous tissues (n = 90). b Kaplan–Meier univariate analysis of overall survival in glioblastoma patients with high (above median) versus low (below median) circENTPD7 levels; P < 0.05 [log-rank test]. c The circENTPD7 was examined in glioblastoma tissues < 3 cm (n = 41) and > 3 cm (n = 49). d The circENTPD7 was examined in glioblastoma tissues at I–II stage (n = 34) and III–IV stage (n = 56). Data are presented as mean ± SD, Student’s t test, ***P < 0.001

Fig. 3

circENTPD7 knockdown represses the proliferation and metastasis of glioblastoma cells. a Transfection efficiency si-circENTPD7 into glioblastoma cells (U87, A172) was examined by RT-qPCR. b Clone formation assay demonstrated the clone number in the circENTPD7 knockdown transfection group and the control transfection. c CCK-8 assay showed the inhibition of circENTPD7 knockdown on the proliferation ability. d Transwell assay demonstrated the circENTPD7 knockdown for the invasion of glioblastoma cells comparing to the control transfection. e The statistical results of d. Data are presented as mean ± SD, Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

circENTPD7 targets miR-101-3p as a miRNA sponge. a Correlation analysis of expression of circENTPD7 and miR-101-3p in glioblastoma tissues were analyzed. b miR-101-3p had the complementary sites with the circENTPD7. c The luciferase activity on behalf of the molecular binding of circENTPD7 and miR-101-3p was tested. d CircENTPD7 levels in the overexpressing glioblastoma cells were tested using RT-PCR. e CircENTPD7 negatively regulated expression of miR-101-3p in glioblastoma cells. f Lysates from U87 and A172 cells with circENTPD7 vector were subjected to biotinylation-cirENTPD7 pull down assay, and expression levels of circENTPD7 were measured by RT-qPCR. g miR-101-3p were measured by RT-qPCR in f. Data are presented as mean ± SD, Student’s t test, ***P < 0.001

Fig. 5

ROS1 serves as the target of miR-101-3p. a Diagram of 3′UTR of ROS1 containing wild type and mutations binding sites of miR-101-3p. b The expression of ROS1 in the glioblastoma were examined. c Expression of miR-101-3p mimics decreased luciferase activities in the cells transfected with plasmids containing wild type 3′UTR of ROS1, while expression of circENTPD7 increased luciferase activities in that; miR-101-3p_mut had no effect on the luciferase activity of ROS1 3′UTR. d miR-101-3p and circENTPD7 had no effect on the luciferase activity of ROS1 3′UTR_Mut; miR-101-3p_mut had no effect on the luciferase activity of circENTPD7. e Expression of miR-101-3p mimics decreased protein levels of ROS1 in glioblastoma cells. f Expression of circENTPD7 increased protein levels of ROS1 in glioblastoma cells. g Transwell assay demonstrated ROS1 was essential  miR-101-3p inhibited cell migration. h Transwell assay demonstrated ROS1 was essential for circENTPD7 induced cell migration. Data are presented as mean ± SD, Student’s t test, **P < 0.01; ***P < 0.001

Fig. 6

knockdown circENTPD7 inhibited tumor growth in vivo. a Representative picture showing excised tumors from circENTPD7 knockdown group (sh-circENTPD7) were much smaller than the negative control group. b Tumor growth curves of the excised tumors were shown. c Tumor weight of the excised tumors were shown. d Expression of miR-101-3p was much higher in sh-circENTPD7 group than in the control group measured by RT-qPCR. e Expression of ROS1 was much lower in sh-circENTPD7 group than in the control group measured by western blot. Representative images of western blot were shown. Data are presented as mean ± SD, Student’s t test, *P < 0.05, **P < 0.01