MicroRNA-377 inhibited proliferation and invasion of human glioblastoma cells by directly targeting specificity protein 1

Abstract

Background: Increasing evidence has indicated that microRNAs (miRNAs) are strongly implicated in the initiation and progression of glioblastoma multiforme (GBM). Here, we identified a novel tumor suppressive miRNA, miR-377, and investigated its role and therapeutic effect for GBM.

Methods: MiRNA global screening was performed on GBM patient samples and adjacent nontumor brain tissues. The expression of miR-377 was detected by real-time reverse-transcription PCR. The effects of miR-377 on GBM cell proliferation, cell cycle progression, invasion, and orthotopic tumorigenicity were investigated The therapeutic effect of miR-377 mimic was explored in a subcutaneous GBM model. Western blot and luciferase reporter assay were used to identify the direct and functional target of miR-377.

Results: MiR-377 was markedly downregulated in human GBM tissues and cell lines. Overexpression of miR-377 dramatically inhibited cell growth both in culture and in orthotopic xenograft tumor models, blocked G1/S transition, and suppressed cell invasion in GBM cells. Importantly, introduction of miR-377 could strongly inhibit tumor growth in a subcutaneous GBM model. Subsequent investigation revealed that specificity protein 1 (Sp1) was a direct and functional target of miR-377 in GBM cells. Silencing of Sp1 recapitulated the antiproliferative and anti-invasive effects of miR-377, whereas restoring the Sp1 expression antagonized the tumor-suppressive function of miR-377. Finally, analysis of miR-377 and Sp1 levels in human GBM tissues revealed that miR-377 is inversely correlated with Sp1 expression.

Conclusion: These findings reveal that miR-377/Sp1 signaling that may be required for GBM development and may consequently serve as a therapeutic target for the treatment of GBM.

Keywords: Sp1; glioblastoma multiforme; invasion; miR-377; proliferation.

Fig. 1.

Expression of miR-377 in GBM samples and cell lines. (A) miRNA profiling of GFAP+ cells isolated from GBM patients and adjacent nontumor glial brain tissues. The pseudocolor represents the intensity scale of tumor versus adjacent nontumor brain tissue. (B) qRT-PCR analysis of miR-377 expression in 107 GBM specimens and 10 normal brain tissues (NBTs). Transcript levels were normalized by U6 expression. Alteration of expression is shown as box plot presentations. ***P < .001. (C) Expression levels of miR-377 in normal human astrocytes (NHAs), 5 glioma cells (U87, U251, LN229, A172 and U118). Transcript levels were normalized by U6 expression. **P < .01. (D) The expression level was categorized as low expression (final score < 0.3) and high expression (final score > 0.3. Correlation between miR-377 levels and median overall survival by Kaplan-Meier analysis of GBM patients with high (n = 47) or low (n = 60) miR-377 expression (P < .001).

Fig. 2.

miR-377 inhibits U87 glioma cell proliferation and induces G0/G1 phase arrest. (A) CCK-8 assay performed in U87 or U251 cells transfected with miR-NC or miR-377. **P < .01. (B) Colony formation assay performed in U87 and U251 cells after miR-377 or miR-NC transduction. The experiments were performed 3 times using triplicate samples, and average scores are indicated with error bars on the histogram. **P < .01. Scale bar = 100 µm. (C) The cell cycle distribution of U87 and U251 cells transfected with miR-377 or miR-NC. (D) The effects of miR-377 on cell cycle proteins (Cylin D1, CDK4, CDK6 and Cyclin E) in the G1/S transition were determined by Western blot analysis. β-actin was used as the loading control.

Fig. 3.

Overexpression of miR-377 blocks tumor growth in orthotopic GBM xenografts. (A) T2-weighted MRI imaging of intracranial tumor growth (arrows) at days 7 and 14 in miR-NC- and miR-377-bearing nude mice. (B) MRI-based quantification of tumor volume development in miR-NC- and miR-377-bearing nude mice. **P < .01. Scale bar = 1 mm (C) Survival curve of mice injected intracranially with 2.5 × 10⁵ of miR-NC-infected U87 cells (n = 10) or miR-377-infected U87 cells (n = 10). (D) Representative H&E staining for tumor cytostructure and histological analysis to detect Sp1 and Ki-67 expression in tumors originated from miR-NC and miR-377-infected U87 cells. Scale bar = 1 mm (first panels), 100 µm (second panels), and 400 µm (third and forth panels) (E) qRT-PCR analysis of miR-377 in 2 primary cell lines after ectopic expression of miR-377 or miR-NC. U6 RNA served as the loading control. **P < .01. (F) Representative H&E staining for tumor cytostructure in tumors originated from miR-NC and miR-377-infected primary cell lines. Scale bar = 1 mm (left panels) and 400 µm (right panels).

Fig. 4.

Treatment with miR-377 mimic inhibits tumor growth in a subcutaneous U87 human GBM xenograft model. (A) U87 cells were implanted subcutaneously into nude mice. Treatment was started 10 days after implantation of U87 cells. miR-377 mimic (20 µg) was injected intratumorly into each subcutaneous tumor every other day. Tumor volumes were measured by a Vernier caliper in the indicated days. **P < .01. (B) Subcutaneous tumors derived from U87 cells in the miR-NC- or miR-377 mimic-treated group were weighed after tumors were harvested. **P < .01. (C) Representative images of subcutaneous tumors were displayed. Scale bar = 0.2 mm (D) Western blot analysis of Sp1 in tumors derived from U87 cells after treatment with miR-377 mimic or vector control. β-actin served as the loading control.

Fig. 5.

Sp1 is the direct target of miR-377 and is involved in miR-377-induced tumor-suppression. (A) Predicted miR-377 target sequence in the 3′-UTR of Sp1 (Sp1–3′-UTR-WT) and mutant containing 7 altered nucleotides in the 3′-UTR of Sp1 (Sp1-3′-UTR-mut). (B) qRT-PCR analysis of miR-377 in U87 and U251 cells after ectopic expression of miR-377 or miR-NC. U6 RNA served as the loading control. **P < .01. (C) Western blot analysis of lysates from miR-NC-transfected or miR-377-transfected U87 and U251 cells probed with Sp1 antibody. β-actin served as the loading control. (D) Luciferase assay of the indicated cells transfected with pGL3-Vector, pGL3-Sp1-3′-UTR-WT, or pGL3-Sp1-3′-UTR-mut reporter with miR-377 mimic. **P < .01.

Fig. 6.

Sp1 is involved in miR-377-induced tumor-suppressive effects. (A) Western blot analysis of Sp1 expression in U87 cells after Sp1 overexpression. β-actin served as the loading control. (B) Colony formation assay performed in U87 cells after Sp1 overexpression. **P < .01. (C) Representative H&E staining for tumor cytostructure in tumors originated from Sp1 or control vector-transfected U87 cells. Scale bar = 1 mm (D) Western blot analysis of Sp1 expression in U87 and U251 cells after knockdown of Sp1. β-actin served as the loading control. (E) Colonies grown from U87 and U251 cells transfected with sh-NC or sh-Sp1 were counted. The experiments were performed 3 times using triplicate samples, and average scores are indicated with error bars on the histogram. **P < .01. Scale bar = 100 µm (F) The cell cycle distribution of miR-NC or miR-377-transduced U87 and U251 cells by introducing pEGFP-Sp1 or empty vector pEGFP. (G) The rescue experiment was performed by introducing pEGFP-Sp1 or pEGFP in the presence or absence of ectopic miR-377 or miR-NC expression in U87 cells. Western blot analysis of Sp1, Cyclin D1, MMP-2, and MMP-9 in the indicated cells. β-actin was used as the loading control. (H) Western blot analysis of Sp1, Cyclin D1, MMP-2, and MMP-9 in shNC or shSp1 cells after transfection of anti-NC or anti-miR-377. β-actin was used as the loading control.

Fig. 7.

SP1 is involved in miR-377-induced reduced invasive capability. (A and B) Transwell invasion assay with miR-NC- or miR-377-transduced U87 and U251 cells without transfection or transfected with pEGFP-Sp1 in the presence or absence of mitomycin C. Scale bar = 100 µm (C and D) Invasion of the above cells was quantitatively analyzed. Columns are the average of 3 independent experiments. **P < .01. (E) Western blot analysis of MMP-2 and MMP-9 in the U87 and U251 cells without transfection or transfected with miR-377 or miR-NC. β-actin was used as the loading control. (F) Histological features of brain tumors derived from miR-NC and miR-377-transduced primary invasive cell lines. Scale bar = 200 µm.

Fig. 8.

Histological analysis of Sp1 protein expression in primary GBM tissues. (A) Representative images of weakly positive or strongly positive Sp1 expression in GBM tissue samples. (B) The immunoreactivity of Sp1 protein in GBM tissues displayed a significant inverse correlation with the relative level of miR-377 expression. **P < .001. Scale bar = 100 µm (upper panels) and 400 µm (lower panels).