Circular RNA 0010117 promotes aggressive glioblastoma behavior by regulating the miRNA-6779-5p/SPEN axis

Abstract

Noncoding RNAs (ncRNAs) play important roles in cancer biology, providing potential targets for cancer intervention. As a new class of endogenous noncoding RNAs, circular RNAs (circRNAs) have been recently identified in cell development and function, and certain types of pathological responses contribute to cancer progression, including glioblastoma. However, the potential mechanisms underlying the relationship between circRNAs and glioblastoma progression are still largely unknown. Methods: The expression and roles of circular RNA 0010117 (circ-0010117) were examined in vitro and in vivo. Quantitative RT‒PCR and western blotting were used to measure the expression of circRNA, miRNA, each gene, or related proteins. Cell biology experiments were performed to detect the biological function of circ-0010117 in glioblastoma cell lines. Moreover, bioinformatics analysis, luciferase reporter assays, and functional complementation analysis were carried out to investigate the target genes. Tumorigenesis was also evaluated by xenografting cells into nude mice. In this study, we found that circ-0010117 is downregulated in glioblastoma compared with corresponding paratumoural tissues. Subsequently, we observed that circ-0010117 can regulate aggressiveness in glioblastoma cells through miR-6779-5p. Furthermore, SPEN was verified as a direct target of miR-6779-5p and contributes to the circ-0010117 regulatory network. In addition, we identified that overexpression of circ-0010117 can suppress tumorigenesis in nude mice. Our findings indicate that circular RNA 0010117 promotes the aggressive behavior of glioblastoma by regulating the miRNA-6779-5p/SPEN axis. Our results provide a rationale for the use of circ-0010117 as a novel potential therapeutic target in glioblastoma.

Keywords: Glioblastoma; SPEN; circ-0010117; miR-6779-5p.

Fig. 1

Circ-0010117 is downregulated in glioblastoma. (A) Circ-0010117 mRNA levels in paratumoural and tumor tissues were determined by qRT‒PCR. *P < 0.05 vs. paratumoural tissues. Circ-0010117 expression in U251 (B) and U87 (C) cells. *P < 0.05 vs. NC. (D) Synthesized interfering oligonucleotides targeting circ-0010117 significantly decreased the expression of circ-0010117 in U251 and U87 cell lines. *P < 0.05 vs. Control.

Fig. 2

Circ-0010117 modulates aggressiveness in glioblastoma. The effect of shcirc-0010117 on the proliferation of U251 (A) and U87 (B) cells was detected with a CCK-8 assay. *P < 0.05 vs. NC. The effect of circ-0010117 overexpression on the proliferation of U251 (C) and U87 (D) cells was detected with a CCK-8 assay. *P < 0.05 vs. NC. The effect of shcirc-0010117 on colony formation of U251 and U87 cells (E, F) was detected by a colony formation assay. *P < 0.05 vs. NC. The effect of circ-0010117 overexpression on colony formation of U251 and U87 cells (G, H) was detected by a colony formation assay. *P < 0.05 vs. NC. The effect of shcirc-0010117 on wound healing of U251 and U87 cells (I). *P < 0.05 vs. NC. The effect of circ-0010117 overexpression on wound healing of U251 and U87 cells (J). *P < 0.05 vs. NC. The effect of shcirc-0010117 on the invasion of U251 and U87 cells (K) was detected by transwell assay. *P < 0.05 vs. NC. The effect of circ-0010117 overexpression on the invasion of U251 and U87 cells (L) was detected by transwell assay. *P < 0.05 vs. NC. The effect of shcirc-0010117 on the apoptosis of U251 and U87 cells (M) was detected by flow cytometry. *P < 0.05 vs. NC. The effect of circ-0010117 overexpression on the apoptosis of U251 and U87 cells (N) was detected by flow cytometry. *P < 0.05 vs. NC.

Fig. 3

Circ-0010117 regulates aggressiveness via miRNA-6779-5p. MiR-6779-5p significantly reduced the luciferase activities of circ-0010117 WT but not Mut in U251 (A) and U87 (B) cells. *P < 0.05 vs. NC mimics. (B) The miR-6779-5p expression level was confirmed after inhibiting or overexpressing miR-677-5p. *P < 0.05 vs. NC inhibitor or NC mimics. (C) The effect of shcirc-0010117 and miR-6779-5p inhibitor on the proliferation of U251 and U87 cells was detected with CCK-8 assays. *P < 0.05 vs. NC. (D) The effect of circ-0010117 and miR-6779-5p overexpression on the proliferation of U251 and U87 cells was detected with CCK-8 assays. *P < 0.05 vs. NC. (E) The effect of shcirc-0010117 and miR-6779-5p inhibitor on the invasion of U251 and U87 cells was detected with a transwell assay. *P < 0.05 vs. NC. (F) The effect of circ-0010117 and miR-6779-5p overexpression on the invasion of U251 and U87 cells was detected with a transwell assay. *P < 0.05 vs. NC. (G) The effect of shcirc-0010117 and miR-6779-5p inhibitor on U251 and U87 cell apoptosis was detected with flow cytometry. *P < 0.05 vs. NC. (H) The effect of circ-0010117 and miR-6779-5p overexpression on the apoptosis of U251 and U87 cells was detected with flow cytometry. *P < 0.05 vs. NC.

Fig. 4

SPEN contributed to miRNA-6779-5p regulation. MiR-6779-5p significantly reduced the luciferase activities of SPEN WT but not Mut in U251 (A) and U87 (B) cells. *P < 0.05 vs. NC mimics. (B) The SPEN expression level was confirmed after overexpressing (C) or inhibiting (D) miR-677-5p. *P < 0.05 vs. NC mimics or NC inhibitor. (D) The effects of miR-6779-5p and SPEN overexpression on the proliferation of U251 and U87 cells were detected with CCK-8 assays (left). The effect of the miR-6779-5p inhibitor and shSPEN on the proliferation of U251 and U87 cells was detected with CCK-8 assays (right). *P < 0.05 vs. NC mimic or NC inhibitor. (E) The effect of the miR-6779-5p inhibitor and shSPEN on the invasion of U251 and U87 cells was detected with a transwell assay. *P < 0.05 vs. NC inhibitor. (F) The effect of miR-6779-5p and SPEN overexpression on the invasion of U251 and U87 cells was detected with a transwell assay. *P < 0.05 vs. NC mimic. (G) The effect of the miR-6779-5p inhibitor and shSPEN on the apoptosis of U251 and U87 cells was detected with flow cytometry. *P < 0.05 vs. NC inhibitor. (H) The effect of miR-6779-5p and SPEN overexpression on the apoptosis of U251 and U87 cells was detected with flow cytometry. *P < 0.05 vs. NC mimic.

Fig. 5

Circ-0010117 upregulation inhibited tumor growth in vivo. (A) circ-0010117 is stably overexpressed in the U251 cell line. (B) Subcutaneously injected into 4-week-old nude mice. The results showed that tumor size (B, C) and weight (D) were remarkably reduced when transplanted with tumor cells overexpressing circ-0010117. *P < 0.05 vs. control. (E) The influence of circ-0010117 overexpression on the caspase pathway was determined by western blotting. *P < 0.05 vs. control.