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HOXB1 Is a Tumor Suppressor Gene Regulated by miR-3175 in Glioma.

Han L, Liu D, Li Z, Tian N, Han Z, Wang G, Fu Y, Guo Z, Zhu Z, Du C, Tian Y - PLoS ONE (2015)

Bottom Line: The HOXB1 gene plays a critical role as an oncogene in diverse tumors.We show that HOXB1 expression is significantly downregulated in glioma tissues and cell lines, and that its expression may be closely associated with the degree of malignancy.Our results suggest that HOXB1 functions as a tumor suppressor, regulated by miR-3175 in glioma.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China.

ABSTRACT
The HOXB1 gene plays a critical role as an oncogene in diverse tumors. However, the functional role of HOXB1 and the mechanism regulating HOXB1 expression in glioma are not fully understood. A preliminary bioinformatics analysis showed that HOXB1 is ectopically expressed in glioma, and that HOXB1 is a possible target of miR-3175. In this study, we investigated the function of HOXB1 and the relationship between HOXB1 and miR-3175 in glioma. We show that HOXB1 expression is significantly downregulated in glioma tissues and cell lines, and that its expression may be closely associated with the degree of malignancy. Reduced HOXB1 expression promoted the proliferation and invasion of glioma cells, and inhibited their apoptosis in vitro, and the downregulation of HOXB1 was also associated with worse survival in glioma patients. More importantly, HOXB1 was shown experimentally to be a direct target of miR-3175 in this study. The downregulated expression of miR-3175 inhibited cell proliferation and invasion, and promoted apoptosis in glioma. The oncogenicity induced by low HOXB1 expression was prevented by an miR-3175 inhibitor in glioma cells. Our results suggest that HOXB1 functions as a tumor suppressor, regulated by miR-3175 in glioma. These results clarify the pathogenesis of glioma and offer a potential target for its treatment.

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HOXB1 is a direct target of miR-3175.(A) Diagram of the seed sequence of the miR-3175-binding site within the wild-type HOXB1 3′-UTR and the design of the mutated HOXB1 3′-UTR sequence. The mutated HOXB1 3′-UTR sequence is labeled in red. (B) Luciferase assay of U87 cells after cotransfection with the wild-type or mutant HOXB1 3′-UTR and miR-3175 mimics or miR-3175 mimics NC. The pRL-TK Renilla luciferase reporter vector was used as the internal control. Luciferase activity = firefly luciferase/Renilla luciferase. (C) HOXB1 mRNA expression is modulated by miR-3175. Glioma cell lines were transfected with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs, and incubated for 72 h. Cells were harvested and assayed using qRT-PCR. (D-E) Western blot analysis of HOXB1 protein levels in glioma cell lines after transfection with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs at 72 h. (F) The correlation between HOXB1 and miR-3175 expression in human glioma tissue specimens was analyzed with Spearman’s rank correlation. All results are representative of three independent experiments, and a statistical analysis is performed (mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001).
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pone.0142387.g005: HOXB1 is a direct target of miR-3175.(A) Diagram of the seed sequence of the miR-3175-binding site within the wild-type HOXB1 3′-UTR and the design of the mutated HOXB1 3′-UTR sequence. The mutated HOXB1 3′-UTR sequence is labeled in red. (B) Luciferase assay of U87 cells after cotransfection with the wild-type or mutant HOXB1 3′-UTR and miR-3175 mimics or miR-3175 mimics NC. The pRL-TK Renilla luciferase reporter vector was used as the internal control. Luciferase activity = firefly luciferase/Renilla luciferase. (C) HOXB1 mRNA expression is modulated by miR-3175. Glioma cell lines were transfected with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs, and incubated for 72 h. Cells were harvested and assayed using qRT-PCR. (D-E) Western blot analysis of HOXB1 protein levels in glioma cell lines after transfection with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs at 72 h. (F) The correlation between HOXB1 and miR-3175 expression in human glioma tissue specimens was analyzed with Spearman’s rank correlation. All results are representative of three independent experiments, and a statistical analysis is performed (mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001).

Mentions: To ascertain the mechanisms by which miR-3175 regulates HOXB1 expression through binding sites in the HOXB1 3′-UTR, as predicted in the bioinformatics analysis (Fig 5A), we synthesized the pmirGLO-WT-HOXB1 and pmirGLO-MUT-HOXB1 plasmids, and performed a HOXB1 3′-UTR luciferase reporter assay in U87 cells. The luciferase activity was significantly reduced in the glioma cells transfected with the miR-3175 mimic and pmirGLO-WT-HOXB1, but no reduction was observed in the cells transfected with pmirGLO-MUT-HOXB1 (Fig 5B). These results suggest that HOXB1 is a direct target of miR-3175. To confirm the relationship between HOXB1 and miR-3175, qRT-PCR and western blot analysis were used to investigate the expression levels of HOXB1 in glioma cell lines 72 h after transfection with miR-3175 mimic, miR-3175 inhibitor, or the corresponding NCs. As shown in Fig 5C–5E, the HOXB1 expression levels in the glioma cell lines were significantly reduced after transfection with the miR-3175 mimic, and increased after transfection with the miR-3175 inhibitor. Spearman’s rank correlation analysis showed that the expression levels of HOXB1 and miR-3175 were inversely correlated in 40 human tissues (Spearman’s R = -0.466; Fig 5F).


HOXB1 Is a Tumor Suppressor Gene Regulated by miR-3175 in Glioma.

Han L, Liu D, Li Z, Tian N, Han Z, Wang G, Fu Y, Guo Z, Zhu Z, Du C, Tian Y - PLoS ONE (2015)

HOXB1 is a direct target of miR-3175.(A) Diagram of the seed sequence of the miR-3175-binding site within the wild-type HOXB1 3′-UTR and the design of the mutated HOXB1 3′-UTR sequence. The mutated HOXB1 3′-UTR sequence is labeled in red. (B) Luciferase assay of U87 cells after cotransfection with the wild-type or mutant HOXB1 3′-UTR and miR-3175 mimics or miR-3175 mimics NC. The pRL-TK Renilla luciferase reporter vector was used as the internal control. Luciferase activity = firefly luciferase/Renilla luciferase. (C) HOXB1 mRNA expression is modulated by miR-3175. Glioma cell lines were transfected with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs, and incubated for 72 h. Cells were harvested and assayed using qRT-PCR. (D-E) Western blot analysis of HOXB1 protein levels in glioma cell lines after transfection with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs at 72 h. (F) The correlation between HOXB1 and miR-3175 expression in human glioma tissue specimens was analyzed with Spearman’s rank correlation. All results are representative of three independent experiments, and a statistical analysis is performed (mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001).
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pone.0142387.g005: HOXB1 is a direct target of miR-3175.(A) Diagram of the seed sequence of the miR-3175-binding site within the wild-type HOXB1 3′-UTR and the design of the mutated HOXB1 3′-UTR sequence. The mutated HOXB1 3′-UTR sequence is labeled in red. (B) Luciferase assay of U87 cells after cotransfection with the wild-type or mutant HOXB1 3′-UTR and miR-3175 mimics or miR-3175 mimics NC. The pRL-TK Renilla luciferase reporter vector was used as the internal control. Luciferase activity = firefly luciferase/Renilla luciferase. (C) HOXB1 mRNA expression is modulated by miR-3175. Glioma cell lines were transfected with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs, and incubated for 72 h. Cells were harvested and assayed using qRT-PCR. (D-E) Western blot analysis of HOXB1 protein levels in glioma cell lines after transfection with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs at 72 h. (F) The correlation between HOXB1 and miR-3175 expression in human glioma tissue specimens was analyzed with Spearman’s rank correlation. All results are representative of three independent experiments, and a statistical analysis is performed (mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001).
Mentions: To ascertain the mechanisms by which miR-3175 regulates HOXB1 expression through binding sites in the HOXB1 3′-UTR, as predicted in the bioinformatics analysis (Fig 5A), we synthesized the pmirGLO-WT-HOXB1 and pmirGLO-MUT-HOXB1 plasmids, and performed a HOXB1 3′-UTR luciferase reporter assay in U87 cells. The luciferase activity was significantly reduced in the glioma cells transfected with the miR-3175 mimic and pmirGLO-WT-HOXB1, but no reduction was observed in the cells transfected with pmirGLO-MUT-HOXB1 (Fig 5B). These results suggest that HOXB1 is a direct target of miR-3175. To confirm the relationship between HOXB1 and miR-3175, qRT-PCR and western blot analysis were used to investigate the expression levels of HOXB1 in glioma cell lines 72 h after transfection with miR-3175 mimic, miR-3175 inhibitor, or the corresponding NCs. As shown in Fig 5C–5E, the HOXB1 expression levels in the glioma cell lines were significantly reduced after transfection with the miR-3175 mimic, and increased after transfection with the miR-3175 inhibitor. Spearman’s rank correlation analysis showed that the expression levels of HOXB1 and miR-3175 were inversely correlated in 40 human tissues (Spearman’s R = -0.466; Fig 5F).

Bottom Line: The HOXB1 gene plays a critical role as an oncogene in diverse tumors.We show that HOXB1 expression is significantly downregulated in glioma tissues and cell lines, and that its expression may be closely associated with the degree of malignancy.Our results suggest that HOXB1 functions as a tumor suppressor, regulated by miR-3175 in glioma.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China.

ABSTRACT
The HOXB1 gene plays a critical role as an oncogene in diverse tumors. However, the functional role of HOXB1 and the mechanism regulating HOXB1 expression in glioma are not fully understood. A preliminary bioinformatics analysis showed that HOXB1 is ectopically expressed in glioma, and that HOXB1 is a possible target of miR-3175. In this study, we investigated the function of HOXB1 and the relationship between HOXB1 and miR-3175 in glioma. We show that HOXB1 expression is significantly downregulated in glioma tissues and cell lines, and that its expression may be closely associated with the degree of malignancy. Reduced HOXB1 expression promoted the proliferation and invasion of glioma cells, and inhibited their apoptosis in vitro, and the downregulation of HOXB1 was also associated with worse survival in glioma patients. More importantly, HOXB1 was shown experimentally to be a direct target of miR-3175 in this study. The downregulated expression of miR-3175 inhibited cell proliferation and invasion, and promoted apoptosis in glioma. The oncogenicity induced by low HOXB1 expression was prevented by an miR-3175 inhibitor in glioma cells. Our results suggest that HOXB1 functions as a tumor suppressor, regulated by miR-3175 in glioma. These results clarify the pathogenesis of glioma and offer a potential target for its treatment.

Show MeSH
Related in: MedlinePlus