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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 the invasion and metastasis of pancreatic cancer cells. (A) Migration and (B) invasion assays were performed using control lentiviral vector-infected or miR-367 vector-infected PANC-1 and BxPC3 cells. The number of cells that passed through the membrane was counted in 10 fields under the × 20 objective lens, original magnification ( × 200). The results obtained from three independent experiments were presented as the mean±s.d. of values. (C) Migration and invasion assays were performed using negative control (anti-miR-C) or anti-miR-367 inhibitor-transfected PANC-1 cells. The results obtained from three independent experiments were presented as the mean±s.d. of values. (D) Impact of miR-367 on tumour metastasis in vivo. Eight weeks after spleen injection of control vector-infected or miR-367 vector-infected cells, the mice were killed, and the livers were dissected. Representative livers from NOD/SCID mice were shown. H&E staining was performed on sections of metastatic tumours and normal liver tissues. Arrows represent the metastatic nodules, original magnification ( × 200). Tumour metastases were quantified by counting the number of metastatic colonies in one histological section of the midportion of liver specimen from mice. The ratio of the metastatic area to the total area was determined in histological sections from the midportion of each liver (7 per group). Student's t-test was used to analyse the statistical significance of the differences between groups.
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fig4: miR-367 promoted the invasion and metastasis of pancreatic cancer cells. (A) Migration and (B) invasion assays were performed using control lentiviral vector-infected or miR-367 vector-infected PANC-1 and BxPC3 cells. The number of cells that passed through the membrane was counted in 10 fields under the × 20 objective lens, original magnification ( × 200). The results obtained from three independent experiments were presented as the mean±s.d. of values. (C) Migration and invasion assays were performed using negative control (anti-miR-C) or anti-miR-367 inhibitor-transfected PANC-1 cells. The results obtained from three independent experiments were presented as the mean±s.d. of values. (D) Impact of miR-367 on tumour metastasis in vivo. Eight weeks after spleen injection of control vector-infected or miR-367 vector-infected cells, the mice were killed, and the livers were dissected. Representative livers from NOD/SCID mice were shown. H&E staining was performed on sections of metastatic tumours and normal liver tissues. Arrows represent the metastatic nodules, original magnification ( × 200). Tumour metastases were quantified by counting the number of metastatic colonies in one histological section of the midportion of liver specimen from mice. The ratio of the metastatic area to the total area was determined in histological sections from the midportion of each liver (7 per group). Student's t-test was used to analyse the statistical significance of the differences between groups.

Mentions: We next evaluated the potential role of miR-367 in the growth of pancreatic cancer cells. We found that miR-367 overexpression had no significant effects on pancreatic cancer cell proliferation in vitro (Supplementary Figure 4A, B). However, miR-367 promoted the migration and invasion of PANC-1 and BxPC3 cells (Figure 4A, B). Furthermore, loss-of-function studies showed that silencing miR-367 with an inhibitor suppressed the migration and invasion of cancer cells (Figure 4C). To further investigate the effects of miR-367 overexpression on PDAC metastasis in vivo, we compared the metastatic nodules formed in the livers of NOD/SCID mice that were injected with tumour cells infected with either a miR-367-expressing lentiviral vector or an empty lentiviral vector. We found that both the control and miR-367-infected cells caused tumours after implanted into the spleen. miR-367 has little effects on the growth of spleen-implanted tumours (Supplementary Figure 4C), although tumours cells infected with miR-367-expressing lentiviral vector downregulated Smad7 expression (Supplementary Figure 4D). However, the mice injected with miR-367-overexpressing cells had increased numbers of liver metastatic colonies and an increased ratio of metastatic area to total area (Figure 4D) compared with mice injected with control cells. We additionally evaluated the effects of the other three miRNAs on cell invasion and found that miR-21 and -106a overexpression promoted PANC1 invasion, whereas miR-181a had no such effects (Supplementary Figure 5A, B). Taken together, these findings suggest that miR-367 promoted pancreatic cancer cell invasion and metastasis.


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 the invasion and metastasis of pancreatic cancer cells. (A) Migration and (B) invasion assays were performed using control lentiviral vector-infected or miR-367 vector-infected PANC-1 and BxPC3 cells. The number of cells that passed through the membrane was counted in 10 fields under the × 20 objective lens, original magnification ( × 200). The results obtained from three independent experiments were presented as the mean±s.d. of values. (C) Migration and invasion assays were performed using negative control (anti-miR-C) or anti-miR-367 inhibitor-transfected PANC-1 cells. The results obtained from three independent experiments were presented as the mean±s.d. of values. (D) Impact of miR-367 on tumour metastasis in vivo. Eight weeks after spleen injection of control vector-infected or miR-367 vector-infected cells, the mice were killed, and the livers were dissected. Representative livers from NOD/SCID mice were shown. H&E staining was performed on sections of metastatic tumours and normal liver tissues. Arrows represent the metastatic nodules, original magnification ( × 200). Tumour metastases were quantified by counting the number of metastatic colonies in one histological section of the midportion of liver specimen from mice. The ratio of the metastatic area to the total area was determined in histological sections from the midportion of each liver (7 per group). Student's t-test was used to analyse the statistical significance of the differences between groups.
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Related In: Results  -  Collection

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fig4: miR-367 promoted the invasion and metastasis of pancreatic cancer cells. (A) Migration and (B) invasion assays were performed using control lentiviral vector-infected or miR-367 vector-infected PANC-1 and BxPC3 cells. The number of cells that passed through the membrane was counted in 10 fields under the × 20 objective lens, original magnification ( × 200). The results obtained from three independent experiments were presented as the mean±s.d. of values. (C) Migration and invasion assays were performed using negative control (anti-miR-C) or anti-miR-367 inhibitor-transfected PANC-1 cells. The results obtained from three independent experiments were presented as the mean±s.d. of values. (D) Impact of miR-367 on tumour metastasis in vivo. Eight weeks after spleen injection of control vector-infected or miR-367 vector-infected cells, the mice were killed, and the livers were dissected. Representative livers from NOD/SCID mice were shown. H&E staining was performed on sections of metastatic tumours and normal liver tissues. Arrows represent the metastatic nodules, original magnification ( × 200). Tumour metastases were quantified by counting the number of metastatic colonies in one histological section of the midportion of liver specimen from mice. The ratio of the metastatic area to the total area was determined in histological sections from the midportion of each liver (7 per group). Student's t-test was used to analyse the statistical significance of the differences between groups.
Mentions: We next evaluated the potential role of miR-367 in the growth of pancreatic cancer cells. We found that miR-367 overexpression had no significant effects on pancreatic cancer cell proliferation in vitro (Supplementary Figure 4A, B). However, miR-367 promoted the migration and invasion of PANC-1 and BxPC3 cells (Figure 4A, B). Furthermore, loss-of-function studies showed that silencing miR-367 with an inhibitor suppressed the migration and invasion of cancer cells (Figure 4C). To further investigate the effects of miR-367 overexpression on PDAC metastasis in vivo, we compared the metastatic nodules formed in the livers of NOD/SCID mice that were injected with tumour cells infected with either a miR-367-expressing lentiviral vector or an empty lentiviral vector. We found that both the control and miR-367-infected cells caused tumours after implanted into the spleen. miR-367 has little effects on the growth of spleen-implanted tumours (Supplementary Figure 4C), although tumours cells infected with miR-367-expressing lentiviral vector downregulated Smad7 expression (Supplementary Figure 4D). However, the mice injected with miR-367-overexpressing cells had increased numbers of liver metastatic colonies and an increased ratio of metastatic area to total area (Figure 4D) compared with mice injected with control cells. We additionally evaluated the effects of the other three miRNAs on cell invasion and found that miR-21 and -106a overexpression promoted PANC1 invasion, whereas miR-181a had no such effects (Supplementary Figure 5A, B). Taken together, these findings suggest that miR-367 promoted pancreatic cancer cell invasion and metastasis.

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