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MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

Ogawa H, Wu X, Kawamoto K, Nishida N, Konno M, Koseki J, Matsui H, Noguchi K, Gotoh N, Yamamoto T, Miyata K, Nishiyama N, Nagano H, Yamamoto H, Obika S, Kataoka K, Doki Y, Mori M, Ishii H - PLoS ONE (2015)

Bottom Line: Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy.This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells.Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; Department of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

ABSTRACT
Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

No MeSH data available.


Related in: MedlinePlus

Expression of miRs in primary tumors and colon cancer cell lines.A) Relative expression of endogenous miR-302s (miR-302a,-b,-c, and-d), miR-369s (miR-369-3p and -5p), and miR-200c in primary tumors and colon cancer cell lines. The human teratocarcinoma cell line NTERA was used as a control. B) miRs expression in HT29, DLD-1 and SW480 cells (n = 3). C) Immunofluorescence analysis of CD31 expression in xenografts from HT29, DLD-1 and SW480 cells. HT29 xenografts exhibited many CD31-positive areas with little necrosis. Scale bars, 200 μm. D) Relative expression of miR-302s and miR-369s in HT29 cells 24 h post-transfection of 30 nM miR-302s plus miR-369s (n = 3). NC, negative control. E) Relative expression of miR-302s in HT29 cells at days 2 and 6 after transfection of miR-302s (n = 3). NC, negative control. F) To evaluate transfection efficiency, a fluorescently labeled dsRNA oligomer was transfected with carbonate apatite (CA) or lipofection (LP) into DLD-1 cells. Transfection efficiency was assessed by fluorescent microscopy and flow cytometry after a single transfection, as indicated. G) Relative expression of miR-302s in HT29 cells 48 h post-transfection by CA or LP (n = 3).
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pone.0127119.g001: Expression of miRs in primary tumors and colon cancer cell lines.A) Relative expression of endogenous miR-302s (miR-302a,-b,-c, and-d), miR-369s (miR-369-3p and -5p), and miR-200c in primary tumors and colon cancer cell lines. The human teratocarcinoma cell line NTERA was used as a control. B) miRs expression in HT29, DLD-1 and SW480 cells (n = 3). C) Immunofluorescence analysis of CD31 expression in xenografts from HT29, DLD-1 and SW480 cells. HT29 xenografts exhibited many CD31-positive areas with little necrosis. Scale bars, 200 μm. D) Relative expression of miR-302s and miR-369s in HT29 cells 24 h post-transfection of 30 nM miR-302s plus miR-369s (n = 3). NC, negative control. E) Relative expression of miR-302s in HT29 cells at days 2 and 6 after transfection of miR-302s (n = 3). NC, negative control. F) To evaluate transfection efficiency, a fluorescently labeled dsRNA oligomer was transfected with carbonate apatite (CA) or lipofection (LP) into DLD-1 cells. Transfection efficiency was assessed by fluorescent microscopy and flow cytometry after a single transfection, as indicated. G) Relative expression of miR-302s in HT29 cells 48 h post-transfection by CA or LP (n = 3).

Mentions: Previous studies indicated that miR-302a,-b,-c, and-d (miR-302s), miR369-3p, -5p (miR-369s) and miR-200c could elicit cellular reprogramming in normal somatic cells [13]. Here, we palneed to reprogram cancer cells using these miRs. We started by investigating the endogenous expression of these miRs in colorectal cancer cell lines and clinical samples of colon cancer (Fig 1A and 1B). Expression of miR-302s in colon cancer was very low or undetectable compared to that in human teratoma NTERA cells, which reportedly express ESC-specific genes and are used as control cells to evaluate ESC-like gene expression in many studies [2, 3, 5, 11]. In contrast, miR-200c expression was high in nine primary tumors examined and many cell lines, such as HT29. Expression levels of miR-302s and miR-369s were lower compared to that of miR-200c, suggesting that miR-200c expression is already high in many colon cancer cells and may be refractory to exogenous overexpression. Next, we transfected only miR-302s or miR-369s in colon cancer cells. To optimize in vivo experiments, which mimic primary tumors, we studied the immunofluorescent staining patterns of CD31, a vascular endothelial marker, using DAPI to counterstain nuclei, in xenografts of HT29, DLD-1 and SW480 cells (Fig 1C). HT29 xenografts had the most abundant vascularization, was uniformly distributed over the whole tumor, and the least amount of necrosis. Further cancer reprogramming analysis by miR-302s and miR-369s was performed mainly with HT29 cells and other supplementary cell lines.


MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

Ogawa H, Wu X, Kawamoto K, Nishida N, Konno M, Koseki J, Matsui H, Noguchi K, Gotoh N, Yamamoto T, Miyata K, Nishiyama N, Nagano H, Yamamoto H, Obika S, Kataoka K, Doki Y, Mori M, Ishii H - PLoS ONE (2015)

Expression of miRs in primary tumors and colon cancer cell lines.A) Relative expression of endogenous miR-302s (miR-302a,-b,-c, and-d), miR-369s (miR-369-3p and -5p), and miR-200c in primary tumors and colon cancer cell lines. The human teratocarcinoma cell line NTERA was used as a control. B) miRs expression in HT29, DLD-1 and SW480 cells (n = 3). C) Immunofluorescence analysis of CD31 expression in xenografts from HT29, DLD-1 and SW480 cells. HT29 xenografts exhibited many CD31-positive areas with little necrosis. Scale bars, 200 μm. D) Relative expression of miR-302s and miR-369s in HT29 cells 24 h post-transfection of 30 nM miR-302s plus miR-369s (n = 3). NC, negative control. E) Relative expression of miR-302s in HT29 cells at days 2 and 6 after transfection of miR-302s (n = 3). NC, negative control. F) To evaluate transfection efficiency, a fluorescently labeled dsRNA oligomer was transfected with carbonate apatite (CA) or lipofection (LP) into DLD-1 cells. Transfection efficiency was assessed by fluorescent microscopy and flow cytometry after a single transfection, as indicated. G) Relative expression of miR-302s in HT29 cells 48 h post-transfection by CA or LP (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4430240&req=5

pone.0127119.g001: Expression of miRs in primary tumors and colon cancer cell lines.A) Relative expression of endogenous miR-302s (miR-302a,-b,-c, and-d), miR-369s (miR-369-3p and -5p), and miR-200c in primary tumors and colon cancer cell lines. The human teratocarcinoma cell line NTERA was used as a control. B) miRs expression in HT29, DLD-1 and SW480 cells (n = 3). C) Immunofluorescence analysis of CD31 expression in xenografts from HT29, DLD-1 and SW480 cells. HT29 xenografts exhibited many CD31-positive areas with little necrosis. Scale bars, 200 μm. D) Relative expression of miR-302s and miR-369s in HT29 cells 24 h post-transfection of 30 nM miR-302s plus miR-369s (n = 3). NC, negative control. E) Relative expression of miR-302s in HT29 cells at days 2 and 6 after transfection of miR-302s (n = 3). NC, negative control. F) To evaluate transfection efficiency, a fluorescently labeled dsRNA oligomer was transfected with carbonate apatite (CA) or lipofection (LP) into DLD-1 cells. Transfection efficiency was assessed by fluorescent microscopy and flow cytometry after a single transfection, as indicated. G) Relative expression of miR-302s in HT29 cells 48 h post-transfection by CA or LP (n = 3).
Mentions: Previous studies indicated that miR-302a,-b,-c, and-d (miR-302s), miR369-3p, -5p (miR-369s) and miR-200c could elicit cellular reprogramming in normal somatic cells [13]. Here, we palneed to reprogram cancer cells using these miRs. We started by investigating the endogenous expression of these miRs in colorectal cancer cell lines and clinical samples of colon cancer (Fig 1A and 1B). Expression of miR-302s in colon cancer was very low or undetectable compared to that in human teratoma NTERA cells, which reportedly express ESC-specific genes and are used as control cells to evaluate ESC-like gene expression in many studies [2, 3, 5, 11]. In contrast, miR-200c expression was high in nine primary tumors examined and many cell lines, such as HT29. Expression levels of miR-302s and miR-369s were lower compared to that of miR-200c, suggesting that miR-200c expression is already high in many colon cancer cells and may be refractory to exogenous overexpression. Next, we transfected only miR-302s or miR-369s in colon cancer cells. To optimize in vivo experiments, which mimic primary tumors, we studied the immunofluorescent staining patterns of CD31, a vascular endothelial marker, using DAPI to counterstain nuclei, in xenografts of HT29, DLD-1 and SW480 cells (Fig 1C). HT29 xenografts had the most abundant vascularization, was uniformly distributed over the whole tumor, and the least amount of necrosis. Further cancer reprogramming analysis by miR-302s and miR-369s was performed mainly with HT29 cells and other supplementary cell lines.

Bottom Line: Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy.This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells.Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; Department of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

ABSTRACT
Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

No MeSH data available.


Related in: MedlinePlus