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. 2015 Sep 29;6(29):27778-93.
doi: 10.18632/oncotarget.4813.

HOXA13 is a potential GBM diagnostic marker and promotes glioma invasion by activating the Wnt and TGF-β pathways

Ran Duan  1   2 , Lei Han  3   4   5   2 , Qixue Wang  3   4   5   2 , Jianwei Wei  3   4   5   2 , Luyue Chen  3   4   5   2 , Jianning Zhang  4   5   2 , Chunsheng Kang  3   4   5   2 , Lei Wang  1   2   6
Affiliations

HOXA13 is a potential GBM diagnostic marker and promotes glioma invasion by activating the Wnt and TGF-β pathways

Ran Duan et al. Oncotarget. .

Abstract

Homeobox (HOX) genes, including HOXA13, are involved in human cancer. We found that HOXA13 expression was associated with glioma grade and prognosis. Bioinformatics analysis revealed that most of the HOXA13-associated genes were enriched in cancer-related signaling pathways and mainly involved in the regulation of transcription. We transfected four glioma cell lines with Lenti-si HOXA13. HOXA13 increased cell proliferation and invasion and inhibited apoptosis. HOXA13 decreased β-catenin, phospho-smad2, and phospho-smad3 in the nucleus and increased phospho-β-catenin in the cytoplasm. Furthermore, downregulation of HOXA13 in orthotopic tumors decreased tumor growth. We suggest that HOXA13 promotes glioma progression in part via Wnt- and TGF-β-induced EMT and is a potential diagnostic biomarker for glioblastoma and an independent prognostic factor in high-grade glioma.

Keywords: HOXA13; Homeobox (HOX) gene; SMAD; epithelial-to-mesenchymal transition (EMT); glioma.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Expression of HOX genes in different solid tumors
The abnormal expression of HOX genes (coding genes) in various solid tumors is shown in respective gene sites. The different colors correspond to nine common solid tumors.
Figure 2
Figure 2. The expression of 5′ HOXA genes is increased in high grade glioma
A. Western blot analysis was used to examine the expression of HOXA9, HOXA10, HOXA11, and HOXA13 in glioma samples. GAPDH was used as a loading control. B. HOXA9, HOXA10, HOXA11, and HOXA13 mRNA expression levels in 64 glioma cases were analyzed by qRT-PCR. The reaction products were electrophoresed through a 3.0% agarose gel that was stained with ethidium bromide and photographed.
Figure 3
Figure 3. HOXA13 mRNA expression is increased in GBM, and high expression of HOXA13 is associated with poor prognoses in some glioma cases
A. The expression levels of HOXA13 were analyzed in glioma tissues of the CGGA, TCGA, Rembrandt, GSE4290 and GSE16011 glioma datasets. B. Kaplan-Meier survival curve analysis of the CGGA, TCGA, Rembrandt and GSE16011 glioma datasets indicated that some HGG patients with lower HOXA13 expression showed prolonged survival compared to patients with high levels of HOXA13.
Figure 4
Figure 4. The HOXA13-associated genes were chiefly enriched in cancer related pathway
A. Correlation analysis performed in the CGGA, TCGA, and Rembrandt glioma samples. HOXA13 associated genes from overlapping CGGA, TCGA and Rembrandt databases were analyzed with KEEG pathway analysis, gene ontology analysis and gene set enrichment analysis (GSEA). B. Enrichment analysis results for pathways analysis. These data were obtained from the KEGG database; the red color corresponds to the up-regulated genes, and the green color corresponds to the down-regulated genes. C. Biological processes enrichment results of two sets of differential genes. This information was retrieved from the GO database. The orders of biological processes listed in the circle are based on their enriched number. D. Correlation analysis and Pathway analysis performed in 301 glioma samples with mRNA expression. A heat map of relative expression of HOXA13-associated genes in glioma tissues sorted by level of HOXA13 expression E. GSEA analysis of gene ontology terms showed that there was enriched expression of gene sets involved in Wnt signaling pathway and cell cycle progression in glioma patients.
Figure 5
Figure 5. HOXA13 siRNA inhibits proliferation, regulates cell cycle progression, and induces apoptosis in GBM cells in vitro
A. Western blot assays of HOXA13 protein expression level of U87 and U87 EGFRvIII in both the nucleus and cytosol after transfection with a Lenti-si HOXA13 construct. GAPDH was used as a loading control. B. HOXA13 expression levels and subcellular location were confirmed using a confocal microscope (1,000×). C. Flow cytometry was performed to examine the cell-cycle in U87, U87 EGFRvIII, LN229, and U251 cells after treatment with Lenti-si HOXA13. The percent of cells in the G0/G1, S, and G2/M phases were measured in four cell lines. D. Annexin V-PI assays indicated greater levels of apoptosis in the Lenti-si HOXA13-treated group compared to the Lenti-NC-treated group. E. Proliferation rates of glioma cells infected with a Lenti-si HOXA13 were measured by MTT assays. All data are represented as the mean +/− SEM; *p < 0.05, **p < 0.01.
Figure 6
Figure 6. The suppression of HOXA13 inhibits invasion via the Wnt and TFG-β pathways in GBM cells
A. Transwell assay was used to examine the invasive ability of U87, U87EGFRvIII, LN229 and U251 cell lines after transfection with Lenti-si HOXA13 and Lenti-NC; B. The expression of Wnt- and TGF-β pathway-associated markers and their distribution in cytoplasm and nucleus were tested after suppressing HOXA13 in four glioma cell lines. C. SMAD2, p-SMAD2, SMAD3 and p-SMAD3 expression and subcellular location were confirmed by confocal microscopy.
Figure 7
Figure 7. The suppression of HOXA13 inhibits tumor growth and is associated with good prognosis in an intracranial glioma murine xenograft model
A. U87EGFRvIII cells pretreated with a lentivirus with HOXA13 siRNA or NC siRNA and a lentivirus containing luciferase were implanted in the brains of nude mice, and tumor formation was assessed by bioluminescence imaging. Changes in the bioluminescent signal were measured at days 7, 14 and 21 after implantation. B. Tissue slices from representative tumors from the two groups were stained with Hematoxylin-eosin-saffron. The images show representative immunohistochemical staining for Ki67 and HOXA13. C. Overall survival was determined by Kaplan-Meier survival curves, and a log-rank test was used to assess the statistical significance of the differences. D. Bioluminescence (BLI) was monitored to assess the tumor growth at days 7, 14 and 21. *P < 0.05.
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