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miR-367 promotes epithelial-to-mesenchymal transition and invasion of pancreatic ductal adenocarcinoma cells by targeting the Smad7-TGF-β signalling pathway.

Zhu Z, Xu Y, Zhao J, Liu Q, Feng W, Fan J, Wang P - Br. J. Cancer (2015)

Bottom Line: However, the potential mechanism underlying aberrant Smad7 expression in human pancreatic ductal adenocarcinoma (PDAC) remains largely unknown.High level of miR-367 expression correlated with poor prognosis of PDAC patients.In addition, we showed that miR-367 promoted epithelial-to-mesenchymal transition by increasing transforming growth factor-β (TGF-β)-induced transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Radiation Oncology, Fudan University Shanghai Cancer Center, 270 Dong An Road, Shanghai 200032, China [2] Department of Oncology, Shanghai Medical College, Fudan University, 130 Dong An Road, Shanghai 200032, China.

ABSTRACT

Background: Aberrant Smad7 expression contributes to the invasion and metastasis of pancreatic cancer cells. However, the potential mechanism underlying aberrant Smad7 expression in human pancreatic ductal adenocarcinoma (PDAC) remains largely unknown.

Methods: Bioinformatic prediction programmes and luciferase reporter assay were used to identify microRNAs regulating Smad7. The association between miR-367 expression and the overall survival of PDAC patients was evaluated by Kaplan-Meier analysis. The effects of miR-367 and Smad7 on the invasion and metastasis of pancreatic cancer cells were investigated both in vitro and in vivo.

Results: We found that miR-367 downregulated Smad7 expression by directly targeting its 3'-UTR in human pancreatic cancer cells. High level of miR-367 expression correlated with poor prognosis of PDAC patients. Functional studies showed that miR-367 promoted pancreatic cancer invasion in vitro and metastasis in vivo through downregulating Smad7. In addition, we showed that miR-367 promoted epithelial-to-mesenchymal transition by increasing transforming growth factor-β (TGF-β)-induced transcriptional activity.

Conclusions: The present study identified and characterised a signalling pathway, the miR-367/Smad7-TGF-β pathway, which is involved in the invasion and metastasis of pancreatic cancer cells. Our results suggest that miR-367 may be a promising therapeutic target for the treatment of human pancreatic cancer.

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miR-367 promoted EMT by increasing TGF-β-induced transcriptional activity. (A) Control lentiviral vector-infected or miR-367 vector-infected PANC-1 cells were uninduced or induced by TGF-β1 (200 pM) for 1 h. Whole-cell extracts were analysed by western blot analysis using antibodies against Smad2, Smad3, Smad4, and phosphorylated Smad2/3. GAPDH was used as a loading control. (B) Control vector-expressing and miR-367-expressing cells were co-transfected with a Smad7-expressing vector that contained the entire coding sequence of Smad7 but lacked the 3′-UTR and a TGF-β responsive luciferase reporter, p3TP. TGF-β-induced luciferase activity was measured. The results obtained from three independent experiments were presented as the mean±s.d. of values. (C) BxPC3 cells were stained to detect E-cadherin and vimentin (green). Cell nuclei were counterstained with DAPI, original magnification ( × 200). The morphology of cells was shown by phase-contrast images (ph.con.). Total protein was extracted, and the expressions of E-cadherin, vimentin, a-SMA, and desmin were evaluated by western blot analysis. GAPDH was used as a loading control.
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fig6: miR-367 promoted EMT by increasing TGF-β-induced transcriptional activity. (A) Control lentiviral vector-infected or miR-367 vector-infected PANC-1 cells were uninduced or induced by TGF-β1 (200 pM) for 1 h. Whole-cell extracts were analysed by western blot analysis using antibodies against Smad2, Smad3, Smad4, and phosphorylated Smad2/3. GAPDH was used as a loading control. (B) Control vector-expressing and miR-367-expressing cells were co-transfected with a Smad7-expressing vector that contained the entire coding sequence of Smad7 but lacked the 3′-UTR and a TGF-β responsive luciferase reporter, p3TP. TGF-β-induced luciferase activity was measured. The results obtained from three independent experiments were presented as the mean±s.d. of values. (C) BxPC3 cells were stained to detect E-cadherin and vimentin (green). Cell nuclei were counterstained with DAPI, original magnification ( × 200). The morphology of cells was shown by phase-contrast images (ph.con.). Total protein was extracted, and the expressions of E-cadherin, vimentin, a-SMA, and desmin were evaluated by western blot analysis. GAPDH was used as a loading control.

Mentions: The TGF-β signalling pathway is involved in cell invasiveness and metastasis by inducing EMT (Wang et al, 2009a). Smad7 is a common inhibitory Smad that inhibits signal transduction triggered by TGF-βs, activins, and BMPs (Hata et al, 1998; Ebisawa et al, 2001; Bai and Cao, 2002; Shi et al, 2004). Therefore, we investigated the effects of miR-367 on TGF-β signalling activity and EMT in PDAC cells. We found that miR-367 overexpression did not significantly alter the expressions of Smad2, Smad3, and Smad4 in PANC1 cells. However, miR-367 overexpression increased phosphorylated Smad2/3 upon TGF-β1 stimulation (Figure 6A). To determine whether increased Smad2/3 phosphorylation induced by miR-367 overexpression increased the promoter activity of TGF-β, PANC1 cells were transfected with TGF-β responsive p3TP-luciferase construct. Transfection with miR-367 in PANC1 cells significantly increased the luciferase activity, whereas transfection with Smad7 without 3′-UTR significantly abrogated the TGF-β signalling activity induced by miR-367 (Figure 6B). EMT, in response to TGF-β1, is phenotypically characterised by downregulation of a number of epithelial markers and upregulation of several mesenchymal markers (Pardali and Moustakas, 2007). In the present study, we found that miR-367 overexpression loosed cell contact, resulted in spindle-shaped morphology, reduced the expression of E-cadherin (an epithelial marker), and increased the expression of vimentin, a-SMA, and desmin (mesenchymal markers). However, transfection of Smad7 without 3′-UTR significantly abrogated the EMT induced by miR-367 (Figure 6C). Therefore, these results suggest that miR-367 promoted EMT by increasing TGF-β-induced transcriptional activity.


miR-367 promotes epithelial-to-mesenchymal transition and invasion of pancreatic ductal adenocarcinoma cells by targeting the Smad7-TGF-β signalling pathway.

Zhu Z, Xu Y, Zhao J, Liu Q, Feng W, Fan J, Wang P - Br. J. Cancer (2015)

miR-367 promoted EMT by increasing TGF-β-induced transcriptional activity. (A) Control lentiviral vector-infected or miR-367 vector-infected PANC-1 cells were uninduced or induced by TGF-β1 (200 pM) for 1 h. Whole-cell extracts were analysed by western blot analysis using antibodies against Smad2, Smad3, Smad4, and phosphorylated Smad2/3. GAPDH was used as a loading control. (B) Control vector-expressing and miR-367-expressing cells were co-transfected with a Smad7-expressing vector that contained the entire coding sequence of Smad7 but lacked the 3′-UTR and a TGF-β responsive luciferase reporter, p3TP. TGF-β-induced luciferase activity was measured. The results obtained from three independent experiments were presented as the mean±s.d. of values. (C) BxPC3 cells were stained to detect E-cadherin and vimentin (green). Cell nuclei were counterstained with DAPI, original magnification ( × 200). The morphology of cells was shown by phase-contrast images (ph.con.). Total protein was extracted, and the expressions of E-cadherin, vimentin, a-SMA, and desmin were evaluated by western blot analysis. GAPDH was used as a loading control.
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fig6: miR-367 promoted EMT by increasing TGF-β-induced transcriptional activity. (A) Control lentiviral vector-infected or miR-367 vector-infected PANC-1 cells were uninduced or induced by TGF-β1 (200 pM) for 1 h. Whole-cell extracts were analysed by western blot analysis using antibodies against Smad2, Smad3, Smad4, and phosphorylated Smad2/3. GAPDH was used as a loading control. (B) Control vector-expressing and miR-367-expressing cells were co-transfected with a Smad7-expressing vector that contained the entire coding sequence of Smad7 but lacked the 3′-UTR and a TGF-β responsive luciferase reporter, p3TP. TGF-β-induced luciferase activity was measured. The results obtained from three independent experiments were presented as the mean±s.d. of values. (C) BxPC3 cells were stained to detect E-cadherin and vimentin (green). Cell nuclei were counterstained with DAPI, original magnification ( × 200). The morphology of cells was shown by phase-contrast images (ph.con.). Total protein was extracted, and the expressions of E-cadherin, vimentin, a-SMA, and desmin were evaluated by western blot analysis. GAPDH was used as a loading control.
Mentions: The TGF-β signalling pathway is involved in cell invasiveness and metastasis by inducing EMT (Wang et al, 2009a). Smad7 is a common inhibitory Smad that inhibits signal transduction triggered by TGF-βs, activins, and BMPs (Hata et al, 1998; Ebisawa et al, 2001; Bai and Cao, 2002; Shi et al, 2004). Therefore, we investigated the effects of miR-367 on TGF-β signalling activity and EMT in PDAC cells. We found that miR-367 overexpression did not significantly alter the expressions of Smad2, Smad3, and Smad4 in PANC1 cells. However, miR-367 overexpression increased phosphorylated Smad2/3 upon TGF-β1 stimulation (Figure 6A). To determine whether increased Smad2/3 phosphorylation induced by miR-367 overexpression increased the promoter activity of TGF-β, PANC1 cells were transfected with TGF-β responsive p3TP-luciferase construct. Transfection with miR-367 in PANC1 cells significantly increased the luciferase activity, whereas transfection with Smad7 without 3′-UTR significantly abrogated the TGF-β signalling activity induced by miR-367 (Figure 6B). EMT, in response to TGF-β1, is phenotypically characterised by downregulation of a number of epithelial markers and upregulation of several mesenchymal markers (Pardali and Moustakas, 2007). In the present study, we found that miR-367 overexpression loosed cell contact, resulted in spindle-shaped morphology, reduced the expression of E-cadherin (an epithelial marker), and increased the expression of vimentin, a-SMA, and desmin (mesenchymal markers). However, transfection of Smad7 without 3′-UTR significantly abrogated the EMT induced by miR-367 (Figure 6C). Therefore, these results suggest that miR-367 promoted EMT by increasing TGF-β-induced transcriptional activity.

Bottom Line: However, the potential mechanism underlying aberrant Smad7 expression in human pancreatic ductal adenocarcinoma (PDAC) remains largely unknown.High level of miR-367 expression correlated with poor prognosis of PDAC patients.In addition, we showed that miR-367 promoted epithelial-to-mesenchymal transition by increasing transforming growth factor-β (TGF-β)-induced transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Radiation Oncology, Fudan University Shanghai Cancer Center, 270 Dong An Road, Shanghai 200032, China [2] Department of Oncology, Shanghai Medical College, Fudan University, 130 Dong An Road, Shanghai 200032, China.

ABSTRACT

Background: Aberrant Smad7 expression contributes to the invasion and metastasis of pancreatic cancer cells. However, the potential mechanism underlying aberrant Smad7 expression in human pancreatic ductal adenocarcinoma (PDAC) remains largely unknown.

Methods: Bioinformatic prediction programmes and luciferase reporter assay were used to identify microRNAs regulating Smad7. The association between miR-367 expression and the overall survival of PDAC patients was evaluated by Kaplan-Meier analysis. The effects of miR-367 and Smad7 on the invasion and metastasis of pancreatic cancer cells were investigated both in vitro and in vivo.

Results: We found that miR-367 downregulated Smad7 expression by directly targeting its 3'-UTR in human pancreatic cancer cells. High level of miR-367 expression correlated with poor prognosis of PDAC patients. Functional studies showed that miR-367 promoted pancreatic cancer invasion in vitro and metastasis in vivo through downregulating Smad7. In addition, we showed that miR-367 promoted epithelial-to-mesenchymal transition by increasing transforming growth factor-β (TGF-β)-induced transcriptional activity.

Conclusions: The present study identified and characterised a signalling pathway, the miR-367/Smad7-TGF-β pathway, which is involved in the invasion and metastasis of pancreatic cancer cells. Our results suggest that miR-367 may be a promising therapeutic target for the treatment of human pancreatic cancer.

Show MeSH
Related in: MedlinePlus