Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter

Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism. Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation...

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Autores principales:	LIANG Guangxia, QIN Xituan, XIE Wei, XIE Youke
Formato:	article
Lenguaje:	ZH
Publicado:	Magazine House of Cancer Research on Prevention and Treatment 2021
Materias:	sulforaphane mir-200c lung cancer stem cells methylation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
Acceso en línea:	https://doaj.org/article/2fde899e7028413397775335f3eb3231
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spelling	oai:doaj.org-article:2fde899e7028413397775335f3eb32312021-11-05T02:22:52ZSulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter1000-857810.3971/j.issn.1000-8578.2021.21.0375https://doaj.org/article/2fde899e7028413397775335f3eb32312021-10-01T00:00:00Zhttp://html.rhhz.net/ZLFZYJ/html/8578.2021.21.0375.htmhttps://doaj.org/toc/1000-8578Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism. Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation experiment was used to detect the ability of tumor sphere formation; Western blot was applied to explore the expression of stemness-related proteins (such as β-catenin, Klf4, c-myc) in lung adenocarcinoma cells before and after SFN treatment; NGS sequencing was used to analyze the effect of SFN on the expression profile of tumor cell miRNAs. qRT-PCR verified the changes in the transcription level of key miRNAs by SFN. Western blot was used to detect the effect of SFN on the expression of DNMTs in tumor cells. We constructed miR-200c promoter-GFP plasmid, and applied IF, methylation PCR and DNA sequencing methods to detect the effect of SFN on the methylation level of tumor spheres and miRNA promoter. Results The miRNAs expression profile of lung adenocarcinoma tumor spheres changed significantly after SFN (5.0μmol/L) treatment, and miRNA-200c increased the most. Compared with the control group, the expression of β-catenin, Klf4, c-myc and Vimentin genes in H460 and A549 cells of SFN-S group decreased, and the protein expression levels of DNMT1 and DNMT3a were also significantly decreased. Compared with the control group, H460 and A549 cells stably expressing pEGFP-R200c plasmid in SFN-S group significantly reduced tumor sphere diameter, while tumor sphere fluorescence intensity increased, and GFP protein expression was up-regulated. There were 9 CpG-rich regions in the miR-200c promoter region in the above-mentioned pEGFP-R200c plasmid cell line, and the methylation levels were 88.9%, 44.4% and 38.8% in the control group, SFN-S group and 5-Aza-dC group, respectively. Conclusion SFN may downregulate the expression of stem-related genes in lung cancer stem cells by epigenetically decreasing the methylation level of miR-200c promoter and promoting the transcription of miR-200c.LIANG GuangxiaQIN XituanXIE WeiXIE YoukeMagazine House of Cancer Research on Prevention and Treatmentarticlesulforaphanemir-200clung cancer stem cellsmethylationNeoplasms. Tumors. Oncology. Including cancer and carcinogensRC254-282ZHZhongliu Fangzhi Yanjiu, Vol 48, Iss 10, Pp 934-940 (2021)
institution	DOAJ
collection	DOAJ
language	ZH
topic	sulforaphane mir-200c lung cancer stem cells methylation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
spellingShingle	sulforaphane mir-200c lung cancer stem cells methylation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282 LIANG Guangxia QIN Xituan XIE Wei XIE Youke Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter
description	Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism. Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation experiment was used to detect the ability of tumor sphere formation; Western blot was applied to explore the expression of stemness-related proteins (such as β-catenin, Klf4, c-myc) in lung adenocarcinoma cells before and after SFN treatment; NGS sequencing was used to analyze the effect of SFN on the expression profile of tumor cell miRNAs. qRT-PCR verified the changes in the transcription level of key miRNAs by SFN. Western blot was used to detect the effect of SFN on the expression of DNMTs in tumor cells. We constructed miR-200c promoter-GFP plasmid, and applied IF, methylation PCR and DNA sequencing methods to detect the effect of SFN on the methylation level of tumor spheres and miRNA promoter. Results The miRNAs expression profile of lung adenocarcinoma tumor spheres changed significantly after SFN (5.0μmol/L) treatment, and miRNA-200c increased the most. Compared with the control group, the expression of β-catenin, Klf4, c-myc and Vimentin genes in H460 and A549 cells of SFN-S group decreased, and the protein expression levels of DNMT1 and DNMT3a were also significantly decreased. Compared with the control group, H460 and A549 cells stably expressing pEGFP-R200c plasmid in SFN-S group significantly reduced tumor sphere diameter, while tumor sphere fluorescence intensity increased, and GFP protein expression was up-regulated. There were 9 CpG-rich regions in the miR-200c promoter region in the above-mentioned pEGFP-R200c plasmid cell line, and the methylation levels were 88.9%, 44.4% and 38.8% in the control group, SFN-S group and 5-Aza-dC group, respectively. Conclusion SFN may downregulate the expression of stem-related genes in lung cancer stem cells by epigenetically decreasing the methylation level of miR-200c promoter and promoting the transcription of miR-200c.
format	article
author	LIANG Guangxia QIN Xituan XIE Wei XIE Youke
author_facet	LIANG Guangxia QIN Xituan XIE Wei XIE Youke
author_sort	LIANG Guangxia
title	Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter
title_short	Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter
title_full	Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter
title_fullStr	Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter
title_full_unstemmed	Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter
title_sort	sulforaphane suppressed stemness characters of lung adenocarcinoma stem cells by down-regulating methylation of mir-200c promoter
publisher	Magazine House of Cancer Research on Prevention and Treatment
publishDate	2021
url	https://doaj.org/article/2fde899e7028413397775335f3eb3231
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Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter

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