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Pancreatic cancer-secreted miR-155 implicates in the conversion from normal fibroblasts to cancer-associated fibroblasts.

Pang W, Su J, Wang Y, Feng H, Dai X, Yuan Y, Chen X, Yao W - Cancer Sci. (2015)

Bottom Line: Microvesicles (MV) have been proved to be an important mediator of intercellular communication and can selectively transport secreted microRNA from a donor cell into a recipient cell.We identified that miR-155 was upregulated in PaC-derived MV and we confirmed that normal fibroblasts could convert into CAF after MV containing miR-155 had been taken up.Targeting the circulating microRNA might be a potential therapy for malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University, Shanghai, China.

No MeSH data available.


Related in: MedlinePlus

Normal fibroblasts (NF) converted into cancer-associated fibroblasts (CAF) after being treated with pancreatic cancer (PaC)-derived microvesicles (MV). (a, b) Western blot analysis of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) protein levels in NF treated with PaC-derived MV. (c, d) Edu proliferation assay analysis in NF and NF treated with PaC-derived MV. (e) Quantitative RT-PCR analysis of miRNA expression levels in NF and NF treated with PaC-derived MV. (f, g) Cell migration analysis in NF and NF treated with PaC-derived MV. *P < 0.05; **P < 0.01; ***P < 0.001.
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fig02: Normal fibroblasts (NF) converted into cancer-associated fibroblasts (CAF) after being treated with pancreatic cancer (PaC)-derived microvesicles (MV). (a, b) Western blot analysis of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) protein levels in NF treated with PaC-derived MV. (c, d) Edu proliferation assay analysis in NF and NF treated with PaC-derived MV. (e) Quantitative RT-PCR analysis of miRNA expression levels in NF and NF treated with PaC-derived MV. (f, g) Cell migration analysis in NF and NF treated with PaC-derived MV. *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: PaC cell media was collected after 2 days of cultivation with BxPC-3 and SW1990, and the MV were separated from the media for further investigation. We found that after separating the PaC cell medium into two parts by sequential centrifugation (the MV and MV-free fractions), a profile of miR-21, miR-25 and miR-155 was primarily localized in the MV fraction, while miR-24, miR-196a and miR-221, were localized in the MV-free fraction (Fig. S1c). Interestingly, miR-155 and miR-25, but not miR-21, were also elevated in the activated fibroblasts. Meanwhile, DiI-labeled MV rapidly entered into the cultured NF after incubation (Fig. S1d). These results implied that miR-155 and miR-25 might be packed in MV and engaged by NF. Afterward, the NF treated with PaC-derived MV were verified to assess whether the features of the NF had efficiently changed. As depicted in Figure 2a and d, the upregulation of α-SMA and FAP protein levels, as well as the proliferation ability, was seen in MV-treated NF. Additionally, the CAF-like environment that was presented after co-cultivation (Fig. 2f,g), and the α-SMA immunostaining of the treated NF by the BXPC-3 and SW1990 MV, also supported this conversion (Fig. S1e). Meanwhile, a similar trend of miRNA alterations between the NF before and after the MV treatment was uncovered (Fig. 2e), which indicated that the PaC-derived MV could induce the formation of CAF-like cells, and that the molecules packed in the MV might be mediators. Furthermore, to exclude the MV contamination from cell culture medium or FBS, we prepared MV-free 10% FBS DMEM by ultracentrifugation, in which the PaC cell lines were cultured, and the MV of PaC cells supernatants were collected after 4 days cultivation. We re-analyzed the alpha-SMA and FAP protein levels, which showed similar results, indicating that contaminations of MV derived from bovines or from DMEM impact little on this NF–CAF conversion (Fig. S1f,g).


Pancreatic cancer-secreted miR-155 implicates in the conversion from normal fibroblasts to cancer-associated fibroblasts.

Pang W, Su J, Wang Y, Feng H, Dai X, Yuan Y, Chen X, Yao W - Cancer Sci. (2015)

Normal fibroblasts (NF) converted into cancer-associated fibroblasts (CAF) after being treated with pancreatic cancer (PaC)-derived microvesicles (MV). (a, b) Western blot analysis of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) protein levels in NF treated with PaC-derived MV. (c, d) Edu proliferation assay analysis in NF and NF treated with PaC-derived MV. (e) Quantitative RT-PCR analysis of miRNA expression levels in NF and NF treated with PaC-derived MV. (f, g) Cell migration analysis in NF and NF treated with PaC-derived MV. *P < 0.05; **P < 0.01; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4638007&req=5

fig02: Normal fibroblasts (NF) converted into cancer-associated fibroblasts (CAF) after being treated with pancreatic cancer (PaC)-derived microvesicles (MV). (a, b) Western blot analysis of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) protein levels in NF treated with PaC-derived MV. (c, d) Edu proliferation assay analysis in NF and NF treated with PaC-derived MV. (e) Quantitative RT-PCR analysis of miRNA expression levels in NF and NF treated with PaC-derived MV. (f, g) Cell migration analysis in NF and NF treated with PaC-derived MV. *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: PaC cell media was collected after 2 days of cultivation with BxPC-3 and SW1990, and the MV were separated from the media for further investigation. We found that after separating the PaC cell medium into two parts by sequential centrifugation (the MV and MV-free fractions), a profile of miR-21, miR-25 and miR-155 was primarily localized in the MV fraction, while miR-24, miR-196a and miR-221, were localized in the MV-free fraction (Fig. S1c). Interestingly, miR-155 and miR-25, but not miR-21, were also elevated in the activated fibroblasts. Meanwhile, DiI-labeled MV rapidly entered into the cultured NF after incubation (Fig. S1d). These results implied that miR-155 and miR-25 might be packed in MV and engaged by NF. Afterward, the NF treated with PaC-derived MV were verified to assess whether the features of the NF had efficiently changed. As depicted in Figure 2a and d, the upregulation of α-SMA and FAP protein levels, as well as the proliferation ability, was seen in MV-treated NF. Additionally, the CAF-like environment that was presented after co-cultivation (Fig. 2f,g), and the α-SMA immunostaining of the treated NF by the BXPC-3 and SW1990 MV, also supported this conversion (Fig. S1e). Meanwhile, a similar trend of miRNA alterations between the NF before and after the MV treatment was uncovered (Fig. 2e), which indicated that the PaC-derived MV could induce the formation of CAF-like cells, and that the molecules packed in the MV might be mediators. Furthermore, to exclude the MV contamination from cell culture medium or FBS, we prepared MV-free 10% FBS DMEM by ultracentrifugation, in which the PaC cell lines were cultured, and the MV of PaC cells supernatants were collected after 4 days cultivation. We re-analyzed the alpha-SMA and FAP protein levels, which showed similar results, indicating that contaminations of MV derived from bovines or from DMEM impact little on this NF–CAF conversion (Fig. S1f,g).

Bottom Line: Microvesicles (MV) have been proved to be an important mediator of intercellular communication and can selectively transport secreted microRNA from a donor cell into a recipient cell.We identified that miR-155 was upregulated in PaC-derived MV and we confirmed that normal fibroblasts could convert into CAF after MV containing miR-155 had been taken up.Targeting the circulating microRNA might be a potential therapy for malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University, Shanghai, China.

No MeSH data available.


Related in: MedlinePlus