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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

SNAI2 contributes to chemoresistance and EMT in GBM cells(A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. *P < 0.05, **P < 0.01.
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Figure 6: SNAI2 contributes to chemoresistance and EMT in GBM cells(A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. *P < 0.05, **P < 0.01.

Mentions: We asked if overexpression of SNAI2 could induce drug resistance and EMT in parental GBM cells. For this purpose, we developed a stably SNAI2 over-expressing U87-pcDNA3.1-SNAI2 subline by transfection with pcDNA3.1-SNAI2 (Supplementary Figure 3). As shown in Figure 6A, B, overexpression of SNAI2 reduced the sensitivity of U87 cells to anticancer drugs and induced a shift in cell morphology from tight cell-cell junctions to loss of cell-to-cell contact. Also, enforced expression of SNAI2 promoted cell invasion in vitro (Figure 6C). Furthermore, the downregulation of epithelial marker E-cadherin and the upregulation of mesenchymal markers ZEB1 and vimentin were observed in pcDNA3.1-SNAI2-transfected U87 cells (Figure 6D).


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)

SNAI2 contributes to chemoresistance and EMT in GBM cells(A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. *P < 0.05, **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496192&req=5

Figure 6: SNAI2 contributes to chemoresistance and EMT in GBM cells(A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. *P < 0.05, **P < 0.01.
Mentions: We asked if overexpression of SNAI2 could induce drug resistance and EMT in parental GBM cells. For this purpose, we developed a stably SNAI2 over-expressing U87-pcDNA3.1-SNAI2 subline by transfection with pcDNA3.1-SNAI2 (Supplementary Figure 3). As shown in Figure 6A, B, overexpression of SNAI2 reduced the sensitivity of U87 cells to anticancer drugs and induced a shift in cell morphology from tight cell-cell junctions to loss of cell-to-cell contact. Also, enforced expression of SNAI2 promoted cell invasion in vitro (Figure 6C). Furthermore, the downregulation of epithelial marker E-cadherin and the upregulation of mesenchymal markers ZEB1 and vimentin were observed in pcDNA3.1-SNAI2-transfected U87 cells (Figure 6D).

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