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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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Bmi1 expression and sphere assay in gastrointestinal cancer cell lines co-cultured with M1- or M2-polarized THP-1 macrophages.(A) Cytokine production profile of M1- and-M2 polarized THP-1 macrophages. (B) qRT-PCR analysis of Bmi1 expression in AGS cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in AGS cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (C) qRT-PCR analysis of Bmi1 expression in HCT116 cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in HCT116 cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (D) Microscopic images of 3D sphere cultured AGS cells co-cultured with macrophages, compared with 3D sphere cultured AGS cells only as a control group. Scale bars, 100um. (E) Microscopic images of 3D sphere cultured HCT116 cells co-cultured with macrophages, compared with 3D sphere cultured HCT116 cells only as a control group. Scale bars, 100um. (F) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in AGS cells (*P < 0.05, *P < 0.05, respectively). (G) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in HCT116 cells (*P < 0.05, **P < 0.01, respectively).
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pone-0081839-g002: Bmi1 expression and sphere assay in gastrointestinal cancer cell lines co-cultured with M1- or M2-polarized THP-1 macrophages.(A) Cytokine production profile of M1- and-M2 polarized THP-1 macrophages. (B) qRT-PCR analysis of Bmi1 expression in AGS cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in AGS cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (C) qRT-PCR analysis of Bmi1 expression in HCT116 cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in HCT116 cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (D) Microscopic images of 3D sphere cultured AGS cells co-cultured with macrophages, compared with 3D sphere cultured AGS cells only as a control group. Scale bars, 100um. (E) Microscopic images of 3D sphere cultured HCT116 cells co-cultured with macrophages, compared with 3D sphere cultured HCT116 cells only as a control group. Scale bars, 100um. (F) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in AGS cells (*P < 0.05, *P < 0.05, respectively). (G) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in HCT116 cells (*P < 0.05, **P < 0.01, respectively).

Mentions: We next performed an in vitro co-culture assay with M1- or M2-polarized THP-1 macrophages to examine if macrophages affect Bmi1 expression in cancer cells and cancer cell functions. As shown previously, THP-1 cells were differentiated into M1- or M2-polarized macrophages by distinct cytokines treatment (Figure 2A)[23]. qRT-PCR analysis revealed that Bmi1 expression was significantly increased in AGS and HCT116 cells co-cultured with both M1- and M2-polarized THP-1 macrophages (Figure 2B, C). Bmi1 is involved in the self-renewal capacity through repression of the INK4a-ARF locus, thus we hypothesized that gastrointestinal cells co-cultured with TAMs may possess the capacity for self-renewal through upregulating Bmi1 expression. To investigate the phenotype of gastrointestinal cells co-cultured with TAMs, we performed a 3D sphere culture grown in serum-free non-adherent culture in gastrointestinal cells co-cultured with M1- or M2-polarized THP-1 macrophages (Figure 2D, E). The sphere formation ability of gastrointestinal cells co-cultured with TAMs was enhanced (Figure 2F, G). These results suggested that TAMs upregulated Bmi1 expression and 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)

Bmi1 expression and sphere assay in gastrointestinal cancer cell lines co-cultured with M1- or M2-polarized THP-1 macrophages.(A) Cytokine production profile of M1- and-M2 polarized THP-1 macrophages. (B) qRT-PCR analysis of Bmi1 expression in AGS cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in AGS cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (C) qRT-PCR analysis of Bmi1 expression in HCT116 cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in HCT116 cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (D) Microscopic images of 3D sphere cultured AGS cells co-cultured with macrophages, compared with 3D sphere cultured AGS cells only as a control group. Scale bars, 100um. (E) Microscopic images of 3D sphere cultured HCT116 cells co-cultured with macrophages, compared with 3D sphere cultured HCT116 cells only as a control group. Scale bars, 100um. (F) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in AGS cells (*P < 0.05, *P < 0.05, respectively). (G) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in HCT116 cells (*P < 0.05, **P < 0.01, respectively).
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Related In: Results  -  Collection

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Show All Figures
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pone-0081839-g002: Bmi1 expression and sphere assay in gastrointestinal cancer cell lines co-cultured with M1- or M2-polarized THP-1 macrophages.(A) Cytokine production profile of M1- and-M2 polarized THP-1 macrophages. (B) qRT-PCR analysis of Bmi1 expression in AGS cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in AGS cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (C) qRT-PCR analysis of Bmi1 expression in HCT116 cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in HCT116 cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (D) Microscopic images of 3D sphere cultured AGS cells co-cultured with macrophages, compared with 3D sphere cultured AGS cells only as a control group. Scale bars, 100um. (E) Microscopic images of 3D sphere cultured HCT116 cells co-cultured with macrophages, compared with 3D sphere cultured HCT116 cells only as a control group. Scale bars, 100um. (F) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in AGS cells (*P < 0.05, *P < 0.05, respectively). (G) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in HCT116 cells (*P < 0.05, **P < 0.01, respectively).
Mentions: We next performed an in vitro co-culture assay with M1- or M2-polarized THP-1 macrophages to examine if macrophages affect Bmi1 expression in cancer cells and cancer cell functions. As shown previously, THP-1 cells were differentiated into M1- or M2-polarized macrophages by distinct cytokines treatment (Figure 2A)[23]. qRT-PCR analysis revealed that Bmi1 expression was significantly increased in AGS and HCT116 cells co-cultured with both M1- and M2-polarized THP-1 macrophages (Figure 2B, C). Bmi1 is involved in the self-renewal capacity through repression of the INK4a-ARF locus, thus we hypothesized that gastrointestinal cells co-cultured with TAMs may possess the capacity for self-renewal through upregulating Bmi1 expression. To investigate the phenotype of gastrointestinal cells co-cultured with TAMs, we performed a 3D sphere culture grown in serum-free non-adherent culture in gastrointestinal cells co-cultured with M1- or M2-polarized THP-1 macrophages (Figure 2D, E). The sphere formation ability of gastrointestinal cells co-cultured with TAMs was enhanced (Figure 2F, G). These results suggested that TAMs upregulated Bmi1 expression and 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