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MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2.

Liao H, Bai Y, Qiu S, Zheng L, Huang L, Liu T, Wang X, Liu Y, Xu N, Yan X, Guo H - Oncotarget (2015)

Bottom Line: We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87).Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance.SNAI2 was identified as a direct target gene of miR-203.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Neurosurgery Institute of Guangdong, Key Laboratory on Brain Function Repair and Regeneration of Guangdong, Zhujiang Hospital, Southern Medical University, Guangzhou, China.

ABSTRACT
Epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration, invasion, and drug resistance in several types of cancer. In this study, our aim was to clarify microRNAs (miRNAs)-related mechanisms underlying EMT followed by acquired resistance to chemotherapy in glioblastoma (GBM). We used multiple methods to achieve our goal including microarray analysis, qRT-PCR, western blotting analysis, loss/gain-of-function analysis, luciferase assays, drug sensitivity assays, wound-healing assay and invasion assay. We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87). Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance. SNAI2 was identified as a direct target gene of miR-203. The knockdown of SNAI2 by short hairpin RNA (shRNA) inhibited EMT and drug resistance. In GBM patients, miR-203 expression was inversely related to SNAI2 expression, and those tumors with low expression of miR-203 experienced poorer clinical outcomes. Our findings indicate that re-expression of miR-203 or targeting SNAI2 might serve as potential therapeutic approaches to overcome chemotherapy resistance in GBM.

No MeSH data available.


Related in: MedlinePlus

Imatinib-resistant U251AR and U87AR cells exhibit EMT characteristics(A) Morphological differences between parental cells and imatinib-resistant GBM cells. Scale bar, 100 μm. (B) Immunofluorescence staining of cell-cell junction protein E-cadherin. Parental and resistant GBM cells were stained with E-cadherin (green) as well as DAPI (blue) and pictures were taken at ×40 magnification. Nuclei are stained in blue with DAPI. (C) The mRNA and protein levels of EMT markers in U251 and U251AR cells were respectively detected by qRT-PCR and western blotting. (D) The mRNA and protein levels of EMT markers in U87 and U87AR cells were respectively detected by qRT-PCR and western blotting. (E) Transwell invasion assay proves an altered invasive behavior of imatinib-resistant GBM cells. Scale bar, 200 μm. (F) The cell viability of parental and resistant GBM cells after treatment with 50 μg/ml TMZ for 24, 48, 72, 96 and 120 h. Data are represented as mean±s.d. of three independent experiments. TMZ, temozolomide. *P < 0.05, **P < 0.001.
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Figure 1: Imatinib-resistant U251AR and U87AR cells exhibit EMT characteristics(A) Morphological differences between parental cells and imatinib-resistant GBM cells. Scale bar, 100 μm. (B) Immunofluorescence staining of cell-cell junction protein E-cadherin. Parental and resistant GBM cells were stained with E-cadherin (green) as well as DAPI (blue) and pictures were taken at ×40 magnification. Nuclei are stained in blue with DAPI. (C) The mRNA and protein levels of EMT markers in U251 and U251AR cells were respectively detected by qRT-PCR and western blotting. (D) The mRNA and protein levels of EMT markers in U87 and U87AR cells were respectively detected by qRT-PCR and western blotting. (E) Transwell invasion assay proves an altered invasive behavior of imatinib-resistant GBM cells. Scale bar, 200 μm. (F) The cell viability of parental and resistant GBM cells after treatment with 50 μg/ml TMZ for 24, 48, 72, 96 and 120 h. Data are represented as mean±s.d. of three independent experiments. TMZ, temozolomide. *P < 0.05, **P < 0.001.

Mentions: By using the parental cell lines U251 and U87, we previously established the imatinib-resistant GBM cell lines U251AR and U87AR [12], which had a cross-resistance to other anticancer drugs (etoposide/VP-16 and temozolomide/TMZ). We investigated whether the acquisition of the multidrug-resistant phenotype was accompanied by morphological changes of the cells. The parent U251 and U87 cells grew in clusters with tight cell-cell junctions, while U251AR and U87AR cells separated from one other and grew as loosely packed spindle-like cells (Figure 1A). This suggested U251AR and U87AR cells had undergone EMT resulting in the acquisition of mesenchymal properties. We next investigated the expression and localization of a key epithelial marker (E-cadherin) in the imatinib-resistant GBM cells compared with their parental cells. E-cadherin predominantly localized at cell-cell contacts in U251 and U87 cells, while the staining intensity was reduced in U251AR and U87AR cells (Figure 1B). Moreover, E-cadherin expression was significantly reduced at the mRNA and protein levels in U251AR and U87AR cells as compared with their parental cells (Figure 1C, D). We also examined the expression of other EMT marker genes by performing gene expression profiling in both U87 and U87AR cells. The gene expression profiling revealed that a range of epithelial marker genes were downregulated and many mesenchymal marker genes were upregulated in U87AR cells (Supplementary Table 1). Additionally, qRT-PCR and western blotting analysis showed that mesenchymal genes ZEB1 and vimentin were also upregulated in U251AR and U87AR cells (Figure 1C, D).


MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2.

Liao H, Bai Y, Qiu S, Zheng L, Huang L, Liu T, Wang X, Liu Y, Xu N, Yan X, Guo H - Oncotarget (2015)

Imatinib-resistant U251AR and U87AR cells exhibit EMT characteristics(A) Morphological differences between parental cells and imatinib-resistant GBM cells. Scale bar, 100 μm. (B) Immunofluorescence staining of cell-cell junction protein E-cadherin. Parental and resistant GBM cells were stained with E-cadherin (green) as well as DAPI (blue) and pictures were taken at ×40 magnification. Nuclei are stained in blue with DAPI. (C) The mRNA and protein levels of EMT markers in U251 and U251AR cells were respectively detected by qRT-PCR and western blotting. (D) The mRNA and protein levels of EMT markers in U87 and U87AR cells were respectively detected by qRT-PCR and western blotting. (E) Transwell invasion assay proves an altered invasive behavior of imatinib-resistant GBM cells. Scale bar, 200 μm. (F) The cell viability of parental and resistant GBM cells after treatment with 50 μg/ml TMZ for 24, 48, 72, 96 and 120 h. Data are represented as mean±s.d. of three independent experiments. TMZ, temozolomide. *P < 0.05, **P < 0.001.
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Figure 1: Imatinib-resistant U251AR and U87AR cells exhibit EMT characteristics(A) Morphological differences between parental cells and imatinib-resistant GBM cells. Scale bar, 100 μm. (B) Immunofluorescence staining of cell-cell junction protein E-cadherin. Parental and resistant GBM cells were stained with E-cadherin (green) as well as DAPI (blue) and pictures were taken at ×40 magnification. Nuclei are stained in blue with DAPI. (C) The mRNA and protein levels of EMT markers in U251 and U251AR cells were respectively detected by qRT-PCR and western blotting. (D) The mRNA and protein levels of EMT markers in U87 and U87AR cells were respectively detected by qRT-PCR and western blotting. (E) Transwell invasion assay proves an altered invasive behavior of imatinib-resistant GBM cells. Scale bar, 200 μm. (F) The cell viability of parental and resistant GBM cells after treatment with 50 μg/ml TMZ for 24, 48, 72, 96 and 120 h. Data are represented as mean±s.d. of three independent experiments. TMZ, temozolomide. *P < 0.05, **P < 0.001.
Mentions: By using the parental cell lines U251 and U87, we previously established the imatinib-resistant GBM cell lines U251AR and U87AR [12], which had a cross-resistance to other anticancer drugs (etoposide/VP-16 and temozolomide/TMZ). We investigated whether the acquisition of the multidrug-resistant phenotype was accompanied by morphological changes of the cells. The parent U251 and U87 cells grew in clusters with tight cell-cell junctions, while U251AR and U87AR cells separated from one other and grew as loosely packed spindle-like cells (Figure 1A). This suggested U251AR and U87AR cells had undergone EMT resulting in the acquisition of mesenchymal properties. We next investigated the expression and localization of a key epithelial marker (E-cadherin) in the imatinib-resistant GBM cells compared with their parental cells. E-cadherin predominantly localized at cell-cell contacts in U251 and U87 cells, while the staining intensity was reduced in U251AR and U87AR cells (Figure 1B). Moreover, E-cadherin expression was significantly reduced at the mRNA and protein levels in U251AR and U87AR cells as compared with their parental cells (Figure 1C, D). We also examined the expression of other EMT marker genes by performing gene expression profiling in both U87 and U87AR cells. The gene expression profiling revealed that a range of epithelial marker genes were downregulated and many mesenchymal marker genes were upregulated in U87AR cells (Supplementary Table 1). Additionally, qRT-PCR and western blotting analysis showed that mesenchymal genes ZEB1 and vimentin were also upregulated in U251AR and U87AR cells (Figure 1C, D).

Bottom Line: We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87).Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance.SNAI2 was identified as a direct target gene of miR-203.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Neurosurgery Institute of Guangdong, Key Laboratory on Brain Function Repair and Regeneration of Guangdong, Zhujiang Hospital, Southern Medical University, Guangzhou, China.

ABSTRACT
Epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration, invasion, and drug resistance in several types of cancer. In this study, our aim was to clarify microRNAs (miRNAs)-related mechanisms underlying EMT followed by acquired resistance to chemotherapy in glioblastoma (GBM). We used multiple methods to achieve our goal including microarray analysis, qRT-PCR, western blotting analysis, loss/gain-of-function analysis, luciferase assays, drug sensitivity assays, wound-healing assay and invasion assay. We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87). Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance. SNAI2 was identified as a direct target gene of miR-203. The knockdown of SNAI2 by short hairpin RNA (shRNA) inhibited EMT and drug resistance. In GBM patients, miR-203 expression was inversely related to SNAI2 expression, and those tumors with low expression of miR-203 experienced poorer clinical outcomes. Our findings indicate that re-expression of miR-203 or targeting SNAI2 might serve as potential therapeutic approaches to overcome chemotherapy resistance in GBM.

No MeSH data available.


Related in: MedlinePlus