Sevoflurane inhibits cell proliferation and migration of glioma by targeting the miR‑27b/VEGF axis

Abstract

Poor prognosis in patients with glioma is primarily due to rapid tumor growth and cell invasion and migration. In addition, microRNA (miR)‑27b is decreased in metastatic glioma. The present study investigated whether sevoflurane inhibited glioma cell progression by targeting miR‑27b. Cell proliferation was analyzed using a Cell Counting Kit‑8 assay and a wound healing assay was used to detect cell migration. Western blotting and reverse transcription‑quantitative PCR analysis were performed to determine the protein and mRNA expression levels. A dual luciferase assay was used to determine the relationship between vascular epithelial growth factor (VEGF) and miR‑27b. VEGF was identified to be a direct target of miR‑27b. Moreover, sevoflurane treatment increased the expression of miR‑27b and decreased the expression of VEGF in U251 and U87 cells. Compared with the control group, sevoflurane inhibited the proliferation and migration of U251 and U87 cells, as well as the expression of matrix metalloproteinase (MMP)‑2 and MMP‑9, which were subsequently abolished by pre‑treatment with an miR‑27b inhibitor. The present results indicated the potential use of sevoflurane by anesthesiologists for the surgical resection of glioma, which may improve patient outcomes in the clinical setting.

Keywords: sevoflurane; cell proliferation; cell migration; glioma.

Figure 1.

Sevoflurane induces the expression of miR-27b in glioma cells. Decreased miR-27b lower expression was identified in (A) U251 cells compared with HEB cells, which was significantly increased by (B) sevoflurane. Similar miR-27b expression levels were found in (C) U87 cells and (D) after sevoflurane treatment. **P<0.01 vs. HEB, or sevoflurane vs. control. miR, microRNA.

Figure 2.

miR-27b mimic and inhibitor affect miR-27b expression levels. In (A) U251 cells, miR-27b was significantly increased after miR-27b mimics transfection, while significantly decreased after (B) miR-27b inhibitor transfection. Similar results were obtained in U87 cells after (C) miR-27b mimic and (D) inhibitor transfection. **P<0.01, ***P<0.001, miR-27b mimics vs. miR-NC mimics; ^##P<0.01, miR-27b inhibitor vs. miR-NC inhibitor. miR, microRNA; NC, negative control.

Figure 3.

VEGF is a target of miR-27b. (A) A conserved binding site for miR-27b in the 3′UTR region of the VEGF gene was identified. miR-27b targeted VEGF 3′UTR in (B) U251 and (C) U87 cells. **P<0.01 miR-27b mimics vs. miR-NC mimics. miR, microRNA; NC, negative control; 3′UTR, 3′ untranslated region; WT, wild-type; VEGF, vascular epithelial growth factor.

Figure 4.

Sevoflurane reduces the expression of VEGF in glioma cells by targeting miR-27b. (A) Increased expression of VEGF in U251 cells compared with HEB cells. The untreated control group acted as a negative control group for the experiments. (B) Compared with control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by miR-27b inhibitor. (C) Increased expression of VEGF in U87 cells compared with HEB cells. (D) Compared with the control group, there was decreased VEGF expression in the sevoflurane group, which was rescued by the miR-27b inhibitor. ^&&P<0.01, U251/U87 vs. HEB. **P<0.01, sevoflurane vs. control. ^#P<0.05 sevoflurane + miR-27b inhibitor vs. sevoflurane. miR, microRNA; VEGF, vascular epithelial growth factor.

Figure 5.

Sevoflurane-induced inhibition of glioma cell proliferation is mediated by the miR-27b/VEGF axis. Compared with the siRNA-NC group, siRNA-1 VEGF and siRNA-2 VEGF significantly decreased the expression of VEGF in (A) U251 and (B) U87 cells. The untreated control group acted as a negative control group for the experiments. The inhibitory effects of sevoflurane on (C) U251 and (D) U87 cell proliferation were abolished by pre-treatment with miRNA-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. ^&P<0.05 siRNA-1 VEGF vs. siRNA-NC. ^&&P<0.01 siRNA-2 VEGF vs. siRNA-NC. **P<0.01 sevoflurane vs. control. ^#P<0.05 sevoflurane + miR-27b inhibitor vs. sevoflurane. n.s, sevoflurane + siRNA VEGF + miR-27b inhibitor vs. sevoflurane. n.s, not significant; siRNA, small interfering RNA; miR, microRNA; VEGF, vascular epithelial growth factor.

Figure 6.

Sevoflurane-induced inhibition of glioma cell migration is mediated by the miR-27b/VEGF axis. The untreated control group acted as a negative control group for the experiments. (A) Images and (B) analysis of the inhibitory effects of sevoflurane on U251 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. (C) Images and (D) analysis of the inhibitory effects of sevoflurane on U87 cell migration were abolished by pre-treatment with miR-27b inhibitor, but were not affected by siRNA VEGF + miR-27b inhibitor. Magnification, ×100. *P<0.05 and **P<0.01 sevoflurane vs. control. ^#P<0.05 sevoflurane + miR-27b inhibitor vs. sevoflurane. n.s, sevoflurane + siRNA VEGF + miR-27b inhibitor vs. sevoflurane. n.s, not significant; siRNA, small interfering RNA; miR, microRNA; VEGF, vascular epithelial growth factor.

Figure 7.

Sevoflurane inhibits the expression levels of MMP-2 and MMP-9. (A) Decreased in protein expression levels of MMP-2 and MMP-9 in U251 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (B) Quantification of western blotting results. (C) Decreased in protein expression levels of MMP-2 and MMP-9 in U87 cells induced by sevoflurane were reversed by pre-treatment with miR-27b inhibitor. However, these were not affected by siRNA VEGF + miR-27b inhibitor. (D) Quantification of western blotting results. **P<0.01 and ***P<0.001 sevoflurane vs. control. ^##P<0.01 sevoflurane + miR-27b inhibitor vs. sevoflurane. n.s, sevoflurane + siRNA VEGF + miR-27b inhibitor vs. sevoflurane. n.s, not significant; siRNA, small interfering RNA; miR, microRNA; VEGF, vascular epithelial growth factor; MMP, matrix metalloproteinase.