Circ_0000215 Increases the Expression of CXCR2 and Promoted the Progression of Glioma Cells by Sponging miR-495-3p

Abstract

Background: In recent years, accumulating studies have found that circular RNA (circRNA) exerts a great effect on tumor progression. Circ_0000215, a novel circRNA, remains largely unknown in terms of its effect and mechanism in glioma.

Method: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to detect the expressions of circ_0000215, miR-495-3p and CXCR2 in human glial cell line HEB and glioma cell lines (A172, U251, U87, SHG-44, LN-18), human glioma tissues and adjacent healthy tissues. Gain- and loss-assays of circ_0000215 were conducted. Cell proliferation ability was detected via the CCK8 assay, and cell invasion ability was examined by Transwell assay. CXCR2 expression was evaluated via RT-PCR and Western blot. Moreover, bioinformatics was applied to analyze the targeting molecules of circ_0000215 and CXCR2. Verification of the relationship between these molecules were supported through the dual-luciferase reporter gene and RNA immunocoprecipitation (RIP) assay.

Results: Circ_0000215 and CXCR2 were remarkably upregulated in glioma tissues and cells. Overexpression of circ_0000215 notably promoted the proliferation, invasion and epithelial-mesenchymal transition (EMT) but inhibited apoptosis of glioma cells, while knocking down circ_0000215 had the opposite effects. Additionally, miR-495-3p, a sponge RNA of circ_0000215, inhibited the growth, invasion and EMT of glioma cells. Mechanistically, miR-495-3p targeted CXCR2 and negatively regulated CXCR2/PI3K/Akt pathway. However, the effects of miR-495-3p were all dampened by overexpression of circ_0000215.

Conclusion: These data demonstrated that circ_0000215 functions as a competitive endogenous RNA by sponging miR-495-3p, thus accelerating glioma progression through CXCR2 axis.

Keywords: CXCR2; circ_0000215; glioma; invasion; miR-495-3p; proliferation.

Figure 1.

The expression of circ _0000215 in glioma tissues and cell lines. A: Circ_0000215 expression in the tissues of glioma patients and normal tissues adjacent to the cancer was measured via qRT-PCR. ***represents P < 0.0001. B. Circ_0000215 expression in normal glioma cell line HEB and glioma cell lines (A172, U251, U87, SHG-44, LN-18) was determined by qRT-PCR. *** P < 0.0001.

Figure 2.

The role of circ _0000215 in glioma cell proliferation, apoptosis and metastasis. A-B. Circ_0000215 overexpression and downexpression model were conducted on A172 (A) and LN-18 (B) cells, and the relative expression of circ_0000215 was determined by qRT-PCR. C-D: The effect of circ_0000215 on cell proliferation was determined via CCK-8. E and F. Cell apoptosis of A-172 and LN-18 cells were detected by flow cytometry. G. Apoptosis related proteins including Bax, Bcl2 and Caspase3 was detected by western blot. H: The effect of circ_0000215 on the invasion ability of glioma cells was determined by Transwell assay. I. Western blot was used to detect EMT markers (E-cadherin, N-cadherin and Vimentin). *,**,*** represent P < 0.05, P < 0.01 and P < 0.001 compared with vector or si-NC group respectively.

Figure 3.

Circ _0000215 targeted miR-495-3p in glioma cells. A: Circ_0000215 has a potential binding site for miR-495-3p (predicted by Circular RNA Interactome (https://circinteractome.nia.nih.gov/)). B: The binding relationship between miR-495-3p and circ_0000215 was verified through the luciferase reporter assay. C-D: RIP assay was used to further confirm the targeted relationship of circ_0000215 and miR-495-3p, the enrichment of them in immunoprecipitation of cell lysates were detected by qRT-PCR. E. miR-495-3p expression after transfection with circ_0000215 or si-circ_0000215 was determined via qRT-PCR. F. The expression of miR-495-3p in glioma tissues and normal tissues adjacent to the cancer was measured by qRT-PCR. NS, **,*** represents P > 0.05, P < 0.01 and P < 0.001. G. Linear regression analysis was used to analyze the relationship between circ_0000215 and miR-495-3p expression in glioma tissues. H. miR-495-3p expression in normal glioma cell line HEB and glioma cell lines was determined by qRT-PCR (A172, U251, U87, SHG-44, LN-18), *,**,*** represents P < 0.05, P < 0.01 and P < 0.001 compared with HEB group.

Figure 4.

Circ_0000215/miR-495-3p regulated glioma cell proliferation and metastasis. A-B. A172(A) and LN-18 (B) cells were transfected miR-495-3p mimics or circ_0000215 overexpression plasmids and miR-495-3p inhibitor or si-circ_0000215 vector. The expression of miR-495-3p in the cells was detected via RT-PCR. ** P < 0.01, *** P < 0.001. C and D. Cell proliferation was determined via CCK-8, *,**represent P < 0.05 and P < 0.01 compared with . E-F. Apoptosis related proteins including Bax, Bcl2 and Caspase3 was detected by western blot. G: The effect of circ_0000215/miR-495-3p on the invasion ability of glioma cells was determined by Transwell assay. H-I. Western blot was used to detect EMT markers (E-cadherin, N-cadherin and Vimentin). *,**,*** represents P < 0.05, P < 0.01 and P < 0.001 compared with vector or si-NC group respectively.

Figure 5.

CXCR2 is a functional target of miR-495-3p in glioma cells. A: miR-495-3p contains a potential binding site for CXCR2 as predicted by TargetScan (http://www.targetscan.org/vert_72/). B. RT-PCR was used to detect CXCR2 mRNA level in glioma and normal tissues. C and D. Pearson repression analysis was used to compare the relationships of circ_0000215 and CXCR2 (C), miR-495-3p and CXCR2 (D). E. Dual-luciferase reporter gene assay was used to verify the binding relationship between miR-495-3p and CXCR2. F. Enrichment of CXCR2 in immunoprecipitation of cell lysates were detected by qRT-PCR in RIP assay. G and H: The expression of CXCR2 was detected via qRT-PCR. I and J: The protein level of CXCR2, PI3 K, Akt were detected by western blot. NS, *,**,*** represents P > 0.05, P < 0.05, P < 0.01 and P < 0.001.