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Identification of miR-30e* regulation of Bmi1 expression mediated by tumor-associated macrophages in gastrointestinal cancer.

Sugihara H, Ishimoto T, Watanabe M, Sawayama H, Iwatsuki M, Baba Y, Komohara Y, Takeya M, Baba H - PLoS ONE (2013)

Bottom Line: On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment.Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3' untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues.Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.

ABSTRACT
Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3' untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.

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miR-30e* suppresses Bmi1 expression in gastrointestinal cells.(A) Western blot analysis of Bmi1 expression in 6 gastrointestinal cancer cell lines. (B) Western blot analysis of Bmi1 expression in high Bmi1-expressing AGS cell lines transfected with negative control (NC) and miR-30e* mimics. (C) Western blot analysis of Bmi1 expression in high Bmi1-expressing HCT116 cell lines transfected with NC and miR-30e* mimics. (D) Western blot analysis of Bmi1 expression in low Bmi1-expressing NUGC4 cell lines transfected with NC and miR-30e* inhibitors. (E) Western blot analysis of Bmi1 expression in low Bmi1-expressing COLO201 cell lines transfected with NC and miR-30e* inhibitors.
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pone-0081839-g003: miR-30e* suppresses Bmi1 expression in gastrointestinal cells.(A) Western blot analysis of Bmi1 expression in 6 gastrointestinal cancer cell lines. (B) Western blot analysis of Bmi1 expression in high Bmi1-expressing AGS cell lines transfected with negative control (NC) and miR-30e* mimics. (C) Western blot analysis of Bmi1 expression in high Bmi1-expressing HCT116 cell lines transfected with NC and miR-30e* mimics. (D) Western blot analysis of Bmi1 expression in low Bmi1-expressing NUGC4 cell lines transfected with NC and miR-30e* inhibitors. (E) Western blot analysis of Bmi1 expression in low Bmi1-expressing COLO201 cell lines transfected with NC and miR-30e* inhibitors.

Mentions: To reveal the functional relevance of miR-30e* expression, we examined the Bmi1 expression in the 6 gastrointestinal cancer cell lines by Western blotting (Figure 3A), and analyzed the relationship between miR-30e* and Bmi1 expression in high Bmi1 expressing cancer cell lines (AGS and HCT116) transfected with miR-30e* mimics, and low Bmi1 expressing cancer cell lines (NUGC4 and COLO201) transfected with miR-30e* inhibitors. Western blot analysis revealed significantly reduced Bmi1 protein levels in AGS and HCT116 cells transfected with miR-30e* mimics compared with controls (Figure 3B, C), and increased levels in NUGC4 and COLO201 cells transfected with miR-30e* inhibitors compared with controls (Figure 3D, E). In addition, to investigate the phenotype of cancer cells transfected with miR-30e* mimics, we performed a 3D sphere culture grown in serum-free non-adherent culture in AGS cells transfected with miR-30e* mimics (Figure 4A). The sphere formation ability of AGS cells transfected with miR-30e* mimics was inhibited (Figure 4B), so we confirmed that the downregulation of miR-30e* caused an enhanced sphere formation.


Identification of miR-30e* regulation of Bmi1 expression mediated by tumor-associated macrophages in gastrointestinal cancer.

Sugihara H, Ishimoto T, Watanabe M, Sawayama H, Iwatsuki M, Baba Y, Komohara Y, Takeya M, Baba H - PLoS ONE (2013)

miR-30e* suppresses Bmi1 expression in gastrointestinal cells.(A) Western blot analysis of Bmi1 expression in 6 gastrointestinal cancer cell lines. (B) Western blot analysis of Bmi1 expression in high Bmi1-expressing AGS cell lines transfected with negative control (NC) and miR-30e* mimics. (C) Western blot analysis of Bmi1 expression in high Bmi1-expressing HCT116 cell lines transfected with NC and miR-30e* mimics. (D) Western blot analysis of Bmi1 expression in low Bmi1-expressing NUGC4 cell lines transfected with NC and miR-30e* inhibitors. (E) Western blot analysis of Bmi1 expression in low Bmi1-expressing COLO201 cell lines transfected with NC and miR-30e* inhibitors.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842972&req=5

pone-0081839-g003: miR-30e* suppresses Bmi1 expression in gastrointestinal cells.(A) Western blot analysis of Bmi1 expression in 6 gastrointestinal cancer cell lines. (B) Western blot analysis of Bmi1 expression in high Bmi1-expressing AGS cell lines transfected with negative control (NC) and miR-30e* mimics. (C) Western blot analysis of Bmi1 expression in high Bmi1-expressing HCT116 cell lines transfected with NC and miR-30e* mimics. (D) Western blot analysis of Bmi1 expression in low Bmi1-expressing NUGC4 cell lines transfected with NC and miR-30e* inhibitors. (E) Western blot analysis of Bmi1 expression in low Bmi1-expressing COLO201 cell lines transfected with NC and miR-30e* inhibitors.
Mentions: To reveal the functional relevance of miR-30e* expression, we examined the Bmi1 expression in the 6 gastrointestinal cancer cell lines by Western blotting (Figure 3A), and analyzed the relationship between miR-30e* and Bmi1 expression in high Bmi1 expressing cancer cell lines (AGS and HCT116) transfected with miR-30e* mimics, and low Bmi1 expressing cancer cell lines (NUGC4 and COLO201) transfected with miR-30e* inhibitors. Western blot analysis revealed significantly reduced Bmi1 protein levels in AGS and HCT116 cells transfected with miR-30e* mimics compared with controls (Figure 3B, C), and increased levels in NUGC4 and COLO201 cells transfected with miR-30e* inhibitors compared with controls (Figure 3D, E). In addition, to investigate the phenotype of cancer cells transfected with miR-30e* mimics, we performed a 3D sphere culture grown in serum-free non-adherent culture in AGS cells transfected with miR-30e* mimics (Figure 4A). The sphere formation ability of AGS cells transfected with miR-30e* mimics was inhibited (Figure 4B), so we confirmed that the downregulation of miR-30e* caused an enhanced sphere formation.

Bottom Line: On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment.Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3' untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues.Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.

ABSTRACT
Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3' untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.

Show MeSH
Related in: MedlinePlus