Methylation-mediated silencing of microRNA-211 promotes cell growth and epithelial to mesenchymal transition through activation of the AKT/β-catenin pathway in GBM

Abstract

Aberrant expression of miR-211 has frequently been reported in cancer studies; however, its role in glioblastoma multiforme (GBM) has not been examined in detail. We investigated the function and the underlying mechanism of miR-211 in GBM. We revealed that miR-211 was downregulated in GBM tissues and cell lines. Restoration of miR-211 inhibited GBM cell growth and invasion both in vitro and in vivo. The epithelial to mesenchymal transition (EMT) phenotype was reversed when miR-211 expression was restored. HMGA2 was identified as a down-stream target of miR-211. MiR-211 had an inhibitory effect on AKT/β-catenin signaling, which was reversed by HMGA2 overexpression or miR-211 restoration. In addition, miR-211 was transcriptionally repressed by EZH2-induced H3K27 trimethylation and promoter methylation. Overall, our findings revealed miR-211 as a tumor suppressor in GBM and mir-211 may be a potential therapeutic target for GBM patients.

Keywords: EZH2; HMGA2; epithelial to mesenchymal transition; glioblastoma; miR-211.

Figure 1. miR-211 expression was downregulated in GBM cell lines and tissues

A. miR-211 expression was downregulated in GBM cell lines, when compared with normal brain cells (NBC). B. miR-211 expression was significantly decreased in GBM tissues versus corresponding non-tumor tissues, as determined by RT-PCR. C. An in situ hybridization assay was performed to examine the expression of miR-211 in GBM tissues and non-tumor tissues. D. GBM patients with high miR-211 expression had longer overall survival time than those with low miR-211 expression.

Figure 2. Restoration of miR-211 inhibited GBM cell growth and invasion in vitro

A. Cells were stably transfected with miR-211. B. LV-miR-211 cells grew slower than the LV-control cells. C. LV-miR-211 cells formed less and smaller colonies than the LV-control cells. D. The cell invasion ability was significantly decreased in LV-miR-211 cells.

Figure 3. Restoration of miR-211 decreased cell growth and invasion in vivo

A. and B. LV-miR-211 cells resulted in decreased growth and tumor weight of subcutaneous xenograft tumors in nude mice. C. and D. LV-miR-211 cells established smaller lung and liver metastatic colonies than the mock group.

Figure 4. HMGA2 was a direct target of miR-211

A. HMGA2 wild-type (WT) and mutant 3′-UTR as indicated. B. miR-211 significantly inhibited the luciferase activity of the HMGA2 WT 3′-UTR but not of the mutant in U87 and U251 cells. C. miR-211 reduced the mRNA level of HMGA2 in U87 and U251 cells. D. miR-211 reduced the protein level of HMGA2 in U87 and U251 cells.

Figure 5. miR-211 inhibited the AKT/β-catenin pathway

A. Restoration of miR-211 decreased p-AKT and nuclear β-catenin expression, whereas anti-miR-211 had the opposite effect. B. Restoration of HMGA2 resulted in the recovery of p-AKT and nuclear β-catenin expression that had been weakened through transfection with miR-211. C. Overexpression of miR-211 inhibited nuclear β-catenin expression, while restoration of HMGA2 counteracted the effect.

Figure 6. EZH2 repressed miR-211 expression through H3K27me3

A. Hypermethylation of the miR-211 promoter was detected in GBM cell lines. A schematic distribution of CpG islands is illustrated by the vertical bars. At least two single clones are represented for each sample. Black and white circles represent hypermethylation and hypomethylation, respectively. B. Schematic representation of the miR-211 locus showing the relative positions of the ChIP PCR forward primers H3K27me3 was steadily enriched at the promoter region of miR-211. IgG was used as a control. C. EZH2 downregulation increased the expression of miR-211. D. DZNep treatment increased the expression of miR-211.