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. 2015 Jan 30;6(3):1740-9.
doi: 10.18632/oncotarget.2714.

Down-regulation of miR-223 reverses epithelial-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells

Jia Ma  1   2 , Binbin Fang  3 , Fanpeng Zeng  3 , Cong Ma  3 , Haijie Pang  3 , Long Cheng  4 , Ying Shi  2 , Hui Wang  3 , Bin Yin  1 , Jun Xia  2 , Zhiwei Wang  1
Affiliations

"V体育官网" Down-regulation of miR-223 reverses epithelial-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells

Jia Ma et al. Oncotarget. .

Abstract

Recent studies have demonstrated that acquisition of epithelial-to-mesenchymal transition (EMT) is associated with drug resistance in pancreatic cancer cells; however, the underlying mechanisms are not fully elucidated. Emerging evidence suggests that microRNAs play a crucial role in controlling EMT. The aims of this study were to explore the potential role of miR-223 in governing EMT in gemcitabine-resistant (GR) pancreatic cancer cells. To achieve this goal, real-time reverse transcription-PCR and western blot analysis were used to validate whether GR cells acquired EMT in AsPC-1 and PANC-1 cells. Invasion, migration, and detachment assays were performed to further identify the EMT characteristics in GR cells. The miR-223 inhibitor was used to determine its role in GR-induced EMT. We found that GR cells acquired EMT features, which obtained elongated fibroblastoid morphology, decreased expression of epithelial marker E-cadherin, and up-regulation of mesenchymal markers VSports手机版. Furthermore, we observed that GR cells are associated with high expression of miR-223. Notably, inhibition of miR-223 led to the reversal of EMT phenotype. More importantly, miR-223 governs GR-induced EMT in part due to down-regulation of its target Fbw7 and subsequent upregulation of Notch-1 in pancreatic cancer. Our study implied that down-regulation of miR-223 could be a novel therapy for pancreatic cancer. .

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Gemcitabine-resistant (GR) cells exhibited EMT phenotype
(A) MTT assay was conducted in parental and GR pancreatic cancer cells. *P < 0.05 Parental cells vs GR cells. (B) Cell morphology was observed by microscopy in parental and GR cells. Parental AsPC-1 and PANC-1 cells displayed an epithelioid appearance, whereas their GR cells showed elongated, irregular fibroblastoid morphology. (C-D) Left panel: Invasion assay was performed to measure the invasive capacity in AsPC-1 GR (C) and PANC-1 GR (D) cells. Right panel: Quantitative results are illustrated for left panel. *P < 0.05 vs control.
Figure 2
Figure 2. Gemcitabine-resistant (GR) cells have enhanced motility activity
(A) Left panel: migration assay was performed in parental and GR cells. Right panel: Quantitative results are illustrated for left panel. *P < 0.05 vs control. (B) Wound assays were performed in parental and GR cells. (C) Cell attachment and attachment assays were assessed in parental and GR cells. *P < 0.05 vs control.
Figure 3
Figure 3. Gemcitabine-resistant (GR) cells have EMT marker changes
(A) Real-time RT-PCR assay was conducted to detect the expression of EMT markers in AsPC-1 and AsPC-1 GR cells. *P < 0.05 GR vs control. (B) Left panel: Western blotting analysis was used to detect the expression of Vimentin, E-cadherin, Snail, Slug, ZEB1 and ZEB2 in AsPC-1 and AsPC-1 GR cells. Right panel: Quantitative results are illustrated for left panel. *P < 0.05 vs control. (C) Real-time RT-PCR assay was used to measure the mRNA levels of EMT markers in PANC-1 and PANC-1 GR cells. *P < 0.05 GR vs control. (D) Western blotting analysis was performed to measure the expression of EMT markers in PANC-1 and PANC-1 GR cells. Right panel: Quantitative results are illustrated for left panel. *P < 0.05 vs control.
Figure 4
Figure 4. Gemcitabine-resistant (GR) cells have high expression of miR-223
(A) TaqMan miRNA assay was conducted to detect the expression of miR-223 in parental and GR cells. *P < 0.05 GR vs control. (B) Cell morphology was taken by microscopy in GR cells transfected with miR-223 inhibitor. (C) Real-time RT-PCR analysis was performed to detect the mRNA levels of EMT markers in AsPC-1 GR cells (Top panel) and PANC-1 GR cells (Bottom panel) after miR-223 inhibitor treatment. (D) Left panel: Western blotting analysis was performed to detect the expression of EMT markers in AsPC-1 GR and PANC-1 GR cells after miR-223 inhibitor treatment. Right panel: Quantitative results are illustrated for left panel. *P < 0.05 vs control.
Figure 5
Figure 5. Down-regulation of miR-223 inhibits motility and invasion in gemcitabine-resistant (GR) cells
(A) Wound healing assays were used to detect the motility in GR cells transfected with miR-223 inhibitor. (B) Migration assay (Top panel) and invasion assay (Bottom panel) were conducted in GR cells transfected with miR-223 inhibitor. (C) Quantitative results are illustrated for panel B. *P < 0.05, **P < 0.01 vs control. (D) Cell attachment and detachment assays were measured in GR cells transfected with miR-223 inhibitor. *P < 0.05, **P < 0.01 vs control.
Figure 6
Figure 6. Gemcitabine-resistant (GR) cells have decreased Fbw7 and increased Notch-1 expression
(A) Real-time RT-PCR analysis was performed to detect the mRNA levels of Fbw7 in parental cells and GR cells (Left panel). Western blotting analysis was used to detect the expression of Fbw7 and Notch-1 in GR cells (Right panel). *P < 0.05 GR vs control. (B) Real-time RT-PCR was conducted to detect Fbw7 mRNA level in GR cells after miR-223 inhibitor treatment (Left panel). The expression of Fbw7 and Notch-1 was determined by Western blotting analysis in GR cells treated with miR-223 inhibitor (Right panel). *P < 0.05 vs control. (C) MTT assay was performed in GR cells treated with miR-223 inhibitor. *P < 0.05 vs control.
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