MicroRNA-625 inhibits the proliferation and increases the chemosensitivity of glioma by directly targeting AKT2

Abstract

Glioma is a malignant tumor for which new therapies are needed. Growing evidence has demonstrated that microRNAs (miRNAs) have a major effect on glioma development. Here, we aimed to characterize a novel anti-cancer miRNA, miR-625, by investigating its expression, function, and mechanism of action in glioma progression. The expression of miR-625 and its target mRNA in human glioma tissues and cell lines was assessed by real-time PCR, western blotting, and immunohistochemistry. Functional significance was assessed by examining cell cycle progression, proliferation, apoptosis, and chemosensitivity to temozolomide in vitro, and by examining growth of subcutaneous glioblastoma in a mouse model in vivo. We found that miR-625 expression was significantly lower in human glioma samples and cell lines than in normal brain tissue and human astrocytes. Furthermore, miR-625 overexpression not only suppressed glioma cell proliferation in culture and in the tumor xenograft model but also induced cell cycle arrest and apoptosis. AKT2 was identified as a direct miR-625 target in glioma cell lines, and AKT2 overexpression reversed the suppressive effects of miR-625 in the cell lines and the tumor xenograft model. Finally, we found that the sensitivity of glioma cells to temozolomide was increased by miR-625 overexpression, and this was reversed by concomitant AKT2 expression. In conclusion, our findings suggest that the miR-625-AKT2 axis could be a new prognostic marker and diagnostic target for gliomas.

Keywords: AKT2; Glioma; miR-625; proliferation.

Figure 1

Downregulation of miR-625 in glioma tissues and cell lines. A. qRT-PCR analysis of miR-625 expression in normal brain tissues (NBTs, n = 5) and glioma tissues (n = 26). B. qRT-PCR analysis of miR-625 expression in NBTs (n = 5) and glioma specimens (n = 26) divided according to WHO pathological classification criteria into grade II (n = 7), grade III (n = 7), and grade IV (n = 12). C. FISH analysis of miR-625 expression shows positive correlation with WHO grade in glioma specimens. D. qRT-PCR analysis of miR-625 expression in normal human astrocytes (NHAs) and five glioma cell lines (U87, LN229, U251, A172, U118). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

MiR-625 overexpression induces cell cycle arrest and inhibits glioma cell growth in vitro. A. CCK-8 assay of proliferation of U87 and U251 glioma cell lines transfected with miR-NC or miR-625. B. Colony-forming assays of U87 and U251 cells transfected with miR-NC or miR-625. C. Representative single or merged images of DAPI- and EDU-stained U87 and U251 cells transfected with miR-NC or miR-625. D. Flow cytometric analysis of cell cycle phase of U251 and U87 cells transfected with miR-NC or miR-625. E. Western blot analysis of Cyclin D1, CDK4, and Cyclin E1 in U87 and U251 cells 48 h after transfection with miR-NC or miR-625. GAPDH served as a loading control. **P < 0.01.

Figure 3

AKT2 mRNA is a direct target of miR-625, and AKT2 protein expression is inversely correlated with that of miR-625 in glioma tissues. (A) Predicted miR-625 target sequence in the wild-type (WT) 3’-UTR of AKT2 mRNA and the mutated construct (mut). (B) Levels of miR-625 in U87 and U251 cells after ectopic expression of miR-625 or miR-NC. (C) Luciferase reporter assay of U87 and U251 cells co-transfected with miR-625 mimic and either pmiRNA-AKT2/3’-UTR-WT or pmiRNA-AKT2/3’-UTR-Mut. (D) Western blot analysis of AKT2 protein expression levels in U87 and U251 cells transfected with miR-NC or miR-625. GAPDH served as the loading control. (E, F) Analysis of AKT2 expression in glioblastoma multiforme (GBM) and normal brain tissue (NBT) from the TCGA (E) and GSE16011 (F) datasets. (G) Western blot analysis of AKT2 protein expression in normal human astrocytes (NHAs) and U87, LN229, U251, A172, and U118 glioma cell lines. (H) Western blot analysis of AKT2 protein expression in five NBTs and 26 glioma specimens. Expression levels were normalized to GAPDH levels. P = 0.0033. (I) Spearman’s correlation analysis of AKT2 protein and miR-625 expression levels in human glioma specimens (r = −0.6035, P < 0.001). **P < 0.01.

Figure 4

MiR-625 overexpression suppresses glioma growth in vivo. (A) Tumor growth curves after subcutaneous injection of nude mice with U87 cells stably expressing miR-NC or miR-625 (n = 4). Tumor volumes were measured every 3 d from days 3 to 30. (B-F) Analysis of miR-NC- and miR-625-expressing U87 tumors on day 30 after injection: representative tumor images (B); tumor weights (C); immunohistochemical staining of AKT2, Ki-67, and cleaved caspase 3 (D); immunofluorescent staining of AKT2 protein (E); and western blot analysis of AKT2, with GAPDH as the loading control (F). *P < 0.01, **P < 0.01, ***P < 0.001.

Figure 5

AKT2 overexpression reverses the suppressive effects of miR-625 on glioma cells in vitro. (A) Western blot analysis of AKT2 expression in U87 and U251 cells expressing control (Ctrl) or AKT2 overexpression vectors. GAPDH served as the loading control. (B) Western blot analysis of AKT2 expression in U87 and U251 cells after transfection with control (si-Ctrl) or AKT2-targeting siRNA (si-AKT2). GAPDH served as the loading control. (C) Representative images and quantification of colony-forming assays of control or AKT2-overexpressing U87 and U251 cells. (D) Representative images and quantification of colony-forming assays of U87 and U251 cells transfected with si-AKT2 or si-Ctrl. (E-H) CCK-8 viability assay (E), colony-forming assay (F), EDU proliferation assay (G), and cell cycle distribution assay (H) of U87 and U251 cells transfected with miR-NC, miR-625, or miR-625+AKT2. (I) Western blot analysis of AKT2 and downstream effector proteins Cyclin D1, CDK4, and Cyclin E1 in U87 and U251 cells transfected with miR-NC, miR-625, or miR-625+AKT2. GAPDH was used as the loading control. **P < 0.01.

Figure 6

Glioma cell chemosensitivity to TMZ is increased by miR-625, and AKT2 overexpression partially reverses this effect. A. CCK-8 assay of U87 and U251 cells transfected with miR-NC or miR-625 and cultured with the indicated concentrations of TMZ for 48 h. B. CCK-8 assay of U87 and U251 cells transfected with miR-NC, miR-625, or miR-625+AKT2 and cultured with 100 µM TMZ for the indicated times. C. Flow cytometric Annexin V-FITC/PI apoptosis assay of U87 and U251 cells transfected with miR-NC, miR-625, or miR-625+AKT2 and cultured with 100 µM TMZ for 48 h. D. Western blot analysis of O-6-methylguanine-DNA methyltransferase (MGMT) expression in U251 cells stably expressing miR-NC, miR-625, or miR-625+AKT2 and cultured with 100 μM TMZ. GAPDH was used as the loading control. *P < 0.05, **P < 0.01.