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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

Characteristics of colon cancer cells upon reprogramming induced by miR-302s or miR-302s plus miR-369s.A) Scheme of cancer reprogramming methods by transfection of miR-302s or miR-302s plus miR-369s. B) miR-302s-induced morphological changes in HT29 cells at days 1, 6, and 28. Reprogrammed HT29 have an embryonic stem cells-like appearance. Scale bars, 100 μm. C) Cells stained using alkaline phosphatase (AP) live stain. Scale bars, 100 μm. D) Relative expression of OCT3/4, SOX2 and NANOG compared to AP− cells by qRT-PCR (n = 3). E) Relative expression of miR-302s and miR-369s in AP+, AP−, and NTERA cells. Mean expression of each miR was compared to that in NTERA cells (n = 3). F) Immunofluorescence of pluripotent stem cell markers Oct3/4, Sox2, and Nanog. Nuclei were stained with DAPI. Scale bars, 100 μm (original magnification, ×200).
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pone.0127119.g002: Characteristics of colon cancer cells upon reprogramming induced by miR-302s or miR-302s plus miR-369s.A) Scheme of cancer reprogramming methods by transfection of miR-302s or miR-302s plus miR-369s. B) miR-302s-induced morphological changes in HT29 cells at days 1, 6, and 28. Reprogrammed HT29 have an embryonic stem cells-like appearance. Scale bars, 100 μm. C) Cells stained using alkaline phosphatase (AP) live stain. Scale bars, 100 μm. D) Relative expression of OCT3/4, SOX2 and NANOG compared to AP− cells by qRT-PCR (n = 3). E) Relative expression of miR-302s and miR-369s in AP+, AP−, and NTERA cells. Mean expression of each miR was compared to that in NTERA cells (n = 3). F) Immunofluorescence of pluripotent stem cell markers Oct3/4, Sox2, and Nanog. Nuclei were stained with DAPI. Scale bars, 100 μm (original magnification, ×200).

Mentions: To determine whether cancer cells can be reprogrammed using miRs, we examined the effect of the introduction of exogenous miR-302s with and without miR-369s three times during transfection period. On day 6, the cells were gently trypsinized, transferred onto MEF feeder cells, and cultured for additional 21 days in ES medium with three chemical inhibitors (3i; 0.5 μM A83-01, an ALK4/5/7 receptor inhibitor; 3 μM CHIR99021, a GSK-3β inhibitor; and 0.5 μM PD0325901, a MEK inhibitor) (Fig 2A) [5,13,18]. Round colonies were induced upon transfection of HT29 with miR-302s. These colonies were similar to those of ESCs/iPSCs and apparently different from those of the parent cells (Fig 2B). ESCs/iPSCs are known to be alkaline phosphatase-positive, so we stained the cells using the AP live stain (Fig 2C) to identify and pick colonies that consisted of truly reprogrammed cells [19]. Among the cells forming ES-like colonies, AP+ but not AP- cells showed an increase the expression levels of OCT3/4, SOX2 and NANOG (Fig 2D) and miR-302s (Fig 2E). Cells transfected with miR-302s plus miR-369s or miR-302s alone were AP+ in culture after 28 days of reprogramming. These reprogrammed cells expressed pluripotent marker proteins such as Oct3/4, Sox2, Nanog, and TRA-1-60 (Fig 2F).


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)

Characteristics of colon cancer cells upon reprogramming induced by miR-302s or miR-302s plus miR-369s.A) Scheme of cancer reprogramming methods by transfection of miR-302s or miR-302s plus miR-369s. B) miR-302s-induced morphological changes in HT29 cells at days 1, 6, and 28. Reprogrammed HT29 have an embryonic stem cells-like appearance. Scale bars, 100 μm. C) Cells stained using alkaline phosphatase (AP) live stain. Scale bars, 100 μm. D) Relative expression of OCT3/4, SOX2 and NANOG compared to AP− cells by qRT-PCR (n = 3). E) Relative expression of miR-302s and miR-369s in AP+, AP−, and NTERA cells. Mean expression of each miR was compared to that in NTERA cells (n = 3). F) Immunofluorescence of pluripotent stem cell markers Oct3/4, Sox2, and Nanog. Nuclei were stained with DAPI. Scale bars, 100 μm (original magnification, ×200).
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pone.0127119.g002: Characteristics of colon cancer cells upon reprogramming induced by miR-302s or miR-302s plus miR-369s.A) Scheme of cancer reprogramming methods by transfection of miR-302s or miR-302s plus miR-369s. B) miR-302s-induced morphological changes in HT29 cells at days 1, 6, and 28. Reprogrammed HT29 have an embryonic stem cells-like appearance. Scale bars, 100 μm. C) Cells stained using alkaline phosphatase (AP) live stain. Scale bars, 100 μm. D) Relative expression of OCT3/4, SOX2 and NANOG compared to AP− cells by qRT-PCR (n = 3). E) Relative expression of miR-302s and miR-369s in AP+, AP−, and NTERA cells. Mean expression of each miR was compared to that in NTERA cells (n = 3). F) Immunofluorescence of pluripotent stem cell markers Oct3/4, Sox2, and Nanog. Nuclei were stained with DAPI. Scale bars, 100 μm (original magnification, ×200).
Mentions: To determine whether cancer cells can be reprogrammed using miRs, we examined the effect of the introduction of exogenous miR-302s with and without miR-369s three times during transfection period. On day 6, the cells were gently trypsinized, transferred onto MEF feeder cells, and cultured for additional 21 days in ES medium with three chemical inhibitors (3i; 0.5 μM A83-01, an ALK4/5/7 receptor inhibitor; 3 μM CHIR99021, a GSK-3β inhibitor; and 0.5 μM PD0325901, a MEK inhibitor) (Fig 2A) [5,13,18]. Round colonies were induced upon transfection of HT29 with miR-302s. These colonies were similar to those of ESCs/iPSCs and apparently different from those of the parent cells (Fig 2B). ESCs/iPSCs are known to be alkaline phosphatase-positive, so we stained the cells using the AP live stain (Fig 2C) to identify and pick colonies that consisted of truly reprogrammed cells [19]. Among the cells forming ES-like colonies, AP+ but not AP- cells showed an increase the expression levels of OCT3/4, SOX2 and NANOG (Fig 2D) and miR-302s (Fig 2E). Cells transfected with miR-302s plus miR-369s or miR-302s alone were AP+ in culture after 28 days of reprogramming. These reprogrammed cells expressed pluripotent marker proteins such as Oct3/4, Sox2, Nanog, and TRA-1-60 (Fig 2F).

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