MiR-124 governs glioma growth and angiogenesis and enhances chemosensitivity by targeting R-Ras and N-Ras

Abstract

Background: Glioma is one of the most aggressive and lethal human brain tumors. Accumulating evidence shows that microRNAs play important roles in cancers, including glioma. Previous studies reported that miR-124 levels were downregulated in glioma specimens. Here, we further investigate the potential role of miR-124 in glioma.

Methods: The expression levels of miR-124 were detected in glioma specimens by quantitative reverse transcriptase PCR. The direct targets of miR-124 were identified by bioinformatics analysis and were further validated by immunoblotting and luciferase reporter assay. The effects of miR-124 on glioma cell proliferation and chemosensitivity to temozolomide were analyzed by Cell-Counting Kit 8 assay. Apoptosis was evaluated by fluorescence activated cell sorting analysis. A xenograft model was used to study the effect of miR-124 on tumor growth and angiogenesis.

Results: Expression levels of miR-124 were greatly downregulated in glioma specimens. related Ras viral oncogene homolog (R-Ras) and neuroblastoma Ras viral oncogene homolog (N-Ras) were identified as direct targets of miR-124. MiR-124 inhibited glioma cell growth, invasion, angiogenesis, and tumor growth and increased chemosensitivity to temozolomide treatment by negatively regulating the Ras family and its downstream signaling pathways: phosphatidylinositol-3 kinase/Akt and Raf/extracellular signal-regulated kinase 1/2. Furthermore, overexpression of R-Ras rescued the inhibitory effects of miR-124. Meanwhile, overexpression of R-Ras and N-Ras restored miR-124-inhibited vascular endothelial growth factor (VEGF) transcription activation. In clinical glioma specimens, protein levels of R-Ras and N-Ras were upregulated and inversely correlated with miR-124 expression levels.

Conclusions: Taken together, these results revealed that miR-124 levels in tumor tissues are associated with glioma occurrence, angiogenesis, and chemoresistance and that miR-124 may be used as a new diagnostic marker and therapeutic target for glioma in the future.

Keywords: N-Ras; R-Ras; carcinogenesis; glioma; miR-124.

Fig. 1.

MiR-124 is downregulated in glioma. (A) Expression levels of miR-124 in 6 normal brain tissues and 24 glioma tissues were analyzed by stem-loop qRT-PCR and normalized to the levels of U6. (B) Relative expression levels of miR-124 in normal brain tissues and 3 different grades of glioma samples. According to the pathological classification, the 24 glioma tissues were divided into 3 groups (8 glioma tissues in each group): WHO grade II, grade III, and grade IV. Data represent mean ± SD of 3 replicates. Student's t-test was used to analyze the significant difference among the 3 groups; * and ** indicate significant difference at P < .05 and P < .01, respectively.

Fig. 2.

MiR-124 directly targets related Ras viral oncogene homolog (R-Ras) and neuroblastoma Ras viral oncogene homolog (N-Ras). (A) The sequence of miR-124 binding sites within R-Ras and N-Ras. The reporter constructs of the R-Ras and N-Ras 3′-UTR sequences and the mutated 3′-UTR sequences are shown in the schematic diagram. (B) Luciferase reporter assay was performed to detect the relative luciferase activities of WT and mut R-Ras or N-Ras reporters; * and ** indicate significant difference at P < .05 and P < .01, respectively. (C) Total proteins from U87 and U251 cells overexpressing miR-124 or miR-NC were subjected to immunoblotting to detect R-Ras and N-Ras expression levels. (D) The expression levels of R-Ras and N-Ras in normal brain tissues and glioma specimens were determined by immunoblotting; the fold changes were normalized to GAPDH. (E) Spearman's correlation analysis was used to determine the corrections between the R-Ras/N-Ras expression and miR-124 levels in human glioma specimens. (Spearman's correlation analysis, r = −0.4701, r = −0.4597, respectively.)

Fig. 3.

MiR-124 overexpression regulates Ras signaling; R-Ras and N-Ras have synergistic effects on restoring miR-124–inhibited VEGF transcriptional activation. (A) The expression levels of c-Raf, Akt, ERK1/2, mTOR, and HIF-1α, the downstream molecules of R-Ras and N-Ras, were detected by immunoblotting analysis. (B) VEGF mRNA levels were determined by qRT-PCR, normalized to GAPDH level; * indicates significant difference at P < .05. (C) MiR-124 overexpression inhibits VEGF transcriptional activation in U87 cells. U87 cells were cotransfected with miR-124 or miR-NC, pRL-TK plasmid, pMAP11-VEGF WT reporter, or pMAP11 mut reporter. Luciferase activities were determined by the dual-luciferase reporter assay system and normalized to the miR-NC group. ** indicates significant difference at P < .01. (D) Forced expression of R-Ras and N-Ras restored miR-124–inhibited VEGF transcriptional activation. U87 cells were cotransfected with miR-124 or miR-NC, VEGF reporter, and pRL-TK plasmid, together with pCMV6 vector alone or with pCMV6–R-Ras and/or pCMV6–N-Ras plasmid at 500 ng (1/4 dose) or 1 μg (1/2 dose). The relative luciferase activities of VEGF reporter were assayed 48 h later. ** indicates significant difference compared with control (P < .01); ^## indicates significant difference compared with miR-124 treatment alone (P < .01).

Fig. 4.

Overexpression of R-Ras reverses the inhibitory effects of miR-124. (A) Cells (3 × 10⁵) overexpressing miR-124 or miR-NC were transfected with pCMV6 vector or pCMV6–R-Ras. After 48 h, the expression levels of c-Raf, p-Akt, p-ERK1/2, mTOR, and HIF-1α protein were determined by immunoblotting. (B) Cells were treated as above. After 48 h, the levels of VEGF and GAPDH were analyzed by qRT-PCR; * and ^# indicate P < .05. * indicates significant difference compared with control; ^# indicates significant difference compared with miR-124 treatment alone.

Fig. 5.

MiR-124 increases chemosensitivity of glioma cells to TMZ treatment, and overexpression of R-Ras and N-Ras partially abolished the apoptotic induction effect of miR-124 in the presence of TMZ. (A) Cell proliferation was evaluated in glioma cells stably expressing miR-NC or miR-124, with or without the TMZ treatments at different doses. CCK8 assay was performed 48 h after treatment. * and ** indicate significant difference at P < .05 and P < .01, respectively. (B) The cell proliferation in 100 μM TMZ treatments were tested every 24 h in both U87 and U251 cells overexpressing miR-124 and miR-NC. ** indicates significant difference at P < .01. (C) U87 cells were cotransfected with miR-124 or miR-NC, pCMV6 vector, pCMV6–R-Ras, or pCMV6–N-Ras cDNA plasmid and cultured in 100 μM TMZ, then subjected to apoptosis analysis by flow cytometry 72 h later. (D) U87 cells stably expressing miR-NC or miR-124 were transfected with 2 μg pCMV6 vector, pCMV6–R-Ras, or pCMV6–N-Ras cDNA plasmid and cultured with or without TMZ. After 72 h, the relative caspase-3 activities were determined. ** indicates significant difference at P < .01. (E) U87 cells overexpressing miR-124 and miR-NC were cotransfected with pCMV6 vector, pCMV6–R-Ras, or pCMV6–N-Ras cDNA plasmid with or without TMZ treatment. Cells were lysed and the proteins were collected after 72 h. The expression levels of R-Ras and N-Ras and their downstream molecule c-Raf were determined by immunoblotting. DMSO, dimethyl sulfoxide.

Fig. 6.

MiR-124 overexpression suppresses tumor angiogenesis in vivo. (A) U87 cells stably expressing miR-124 or miR-NC were mixed with Matrigel and injected into both flanks of nude mice. On day 11 after the implantation, mice were euthanized and Matrigel plugs were trimmed out. The photograph shows the representative Matrigel plugs. Scale bar, 2 mm. (B) The hemoglobin levels in the miR-124 group showed nearly 60% reduction compared with the NC group; (C) tissues were formalin fixed, paraffin embedded, and sectioned at 5 μm using antibody against CD31; magnification, 400×. Scale bar, 20 μm. (D) CD31-positive microvessels were counted in 3 different fields per section at 400× magnification; ** indicates significant difference at P < .01.

Fig. 7.

Overexpression of miR-124 inhibits cell growth, invasion, and tumor growth in vivo. (A) U87 cells stably expressing miR-NC or miR-124 were transfected with pCMV6 vector, pCMV6–R-Ras, or pCMV6–N-Ras cDNA plasmid; CCK8 proliferation assay was performed every 24 h. MiR-124 overexpression decreased U87 cell growth, while overexpression of R-Ras or N-Ras restored miR-124–inhibition effect. (B) Transwell invasion assay of U87 overexpressing miR-124 cells with or without R-Ras or N-Ras overexpression. After being fixed, stained, and photographed, the cells in the bottom of the invasion chamber were measured by the absorbance at 570 nm. Scale bar, 20 µm. ** and ^## indicate significant difference compared with control and miR-124 treatment alone, respectively (P < .01). (C) U87 cells stably expressing miR-124 or NC were injected subcutaneously into nude mice. Each treatment group contained 8 tumors, and the tumor volumes were measured after 7 days, when the xenografts were visible. (D) On day 23, xenografts were removed from the nude mice. The photograph shows the representative tumors from each graph. Scale bar, 2 mm. (E) Tumors from 2 groups were weighed and analyzed; ** indicates significant difference at P < .01. (F) The levels of R-Ras and N-Ras from the tumor tissues in 2 groups were analyzed by immunoblotting.