Basic Study
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World J Stem Cells. Mar 26, 2025; 17(3): 99472
Published online Mar 26, 2025. doi: 10.4252/wjsc.v17.i3.99472
DNA methyltransferase 1/miR-342-3p/Forkhead box M1 signaling axis promotes self-renewal in cervical cancer stem-like cells in vitro and nude mice models
Xiao-Zheng Cao, Yao-Feng Zhang, Yu-Wei Song, Lei Yuan, Hui-Li Tang, Jin-Yuan Li, Ye-Bei Qiu, Jia-Zhi Lin, Ying-Xia Ning, Xiao-Yu Wang, Yong Xu, Shao-Qiang Lin
Xiao-Zheng Cao, Lei Yuan, Shao-Qiang Lin, Guangdong Provincial Engineering Research Center for Esophageal Cancer Precise Therapy, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou 510062, Guangdong Province, China
Xiao-Zheng Cao, Yong Xu, Institute of Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, Guangdong Province, China
Yao-Feng Zhang, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong Province, China
Yu-Wei Song, Central Laboratory, The First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China
Hui-Li Tang, Xiao-Yu Wang, Shao-Qiang Lin, Central Laboratory, The Affiliated Shunde Hospital of Jinan University, Foshan 528000, Guangdong Province, China
Jin-Yuan Li, Department of Pelvic Radiotherapy, Meizhou People’s Hospital, Meizhou 514030, Guangdong Province, China
Ye-Bei Qiu, Department of Oncology, The First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China
Jia-Zhi Lin, Ying-Xia Ning, Department of Gynaecology and Obstetrics, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510000, Guangdong Province, China
Co-corresponding authors: Yong Xu and Shao-Qiang Lin.
Author contributions: Cao XZ conducted the experiments and drafted the manuscript; Zhang YF and Yuan L assisted in the study design and data analysis; Song YW and Li JY performed the immunohistochemistry and western blotting of cell and tumor tissue; Tang HL, Qiu YB, and Lin JZ performed the date collection; Ning YX and Wang XY consulted the literature and contributed to revision; Xu Y and Lin SQ contributed equally as co-corresponding authors, whereby Xu Y led the study design, data interpretation, and manuscript preparation, and Lin SQ co-led the experimental design, data analysis, and manuscript editing; All authors approved the final version of the article.
Supported by Guangzhou Basic and Applied Basic Research Foundation, No. 202201010121; Medical Joint Fund of Jinan University, No. YXZY2024014 and No. YXJC2022001; Hospital Achievement Transformation and Cultivation Project, No. ZH201911; the Key Discipline Project of Guangdong Province, No. 2019-GDXK-0016; and the Medical Science and Technology Research Foundation of Guangdong Province, No. B2021145.
Institutional animal care and use committee statement: The study was reviewed and approved by the Committee of Experimental Animal Feeding and Management approved all research assays (Permit No. D2020041).
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Shao-Qiang Lin, MD, PhD, Guangdong Provincial Engineering Research Center for Esophageal Cancer Precise Therapy, The First Affiliated Hospital of Guangdong Pharmaceutical University, No. 19 Nonglinxia Road, Guangzhou 510062, Guangdong Province, China. sqlin123@163.com
Received: July 29, 2024
Revised: October 24, 2024
Accepted: January 2, 2025
Published online: March 26, 2025
Processing time: 234 Days and 23 Hours
Abstract
BACKGROUND

Cervical cancer (CC) stem cell-like cells (CCSLCs), defined by the capacity of differentiation and self-renewal and proliferation, play a significant role in the progression of CC. However, the molecular mechanisms regulating their self-renewal are poorly understood. Therefore, elucidation of the epigenetic mechanisms that drive cancer stem cell self-renewal will enhance our ability to improve the effectiveness of targeted therapies for cancer stem cells.

AIM

To explore how DNA methyltransferase 1 (DNMT1)/miR-342-3p/Forkhead box M1 (FoxM1), which have been shown to have abnormal expression in CCSLCs, and their signaling pathways could stimulate self-renewal-related stemness in CCSLCs.

METHODS

Sphere-forming cells derived from CC cell lines HeLa, SiHa and CaSki served as CCSLCs. Self-renewal-related stemness was identified by determining sphere and colony formation efficiency, CD133 and CD49f protein level, and SRY-box transcription factor 2 and octamer-binding transcription factor 4 mRNA level. The microRNA expression profiles between HeLa cells and HeLa-derived CCSLCs or mRNA expression profiles that HeLa-derived CCSLCs were transfected with or without miR-342-3p mimic were compared using quantitative PCR analysis. The expression levels of DNMT1 mRNA, miR-342-3p, and FoxM1 protein were examined by quantitative real-time PCR and western blotting. In vivo carcinogenicity was assessed using a mouse xenograft model. The functional effects of the DNMT1/miR-342-3p/FoxM1 axis were examined by in vivo and in vitro gain-of-activity and loss-of-activity assessments. Interplay among DNMT1, miR-342-3p, and FoxM1 was tested by methylation-specific PCR and a respective luciferase reporter assay.

RESULTS

CCSLCs derived from the established HeLa cell lines displayed higher self-renewal-related stemness, including enhanced sphere and colony formation efficiency, increased CD133 and CD49f protein level, and heightened transcriptional quantity of stemness-related factors SRY-box transcription factor 2 and octamer-binding transcription factor 4 in vitro as well as a stronger tumorigenic potential in vivo compared to their parental cells. Moreover, quantitative PCR showed that the miR-342-3p level was downregulated in HeLa-derived CCSLCs compared to HeLa cells. Its mimic significantly decreased DNMT1 and FoxM1 mRNA expression levels in CCSLCs. Knockdown of DNMT1 or miR-342-3p mimic transfection suppressed DNMT1 expression, increased miR-342-3p quantity by promoter demethylation, and inhibited CCSLC self-renewal. Inhibition of FoxM1 by shRNA transfection also resulted in the attenuation of CCSLC self-renewal but had little effect on the DNMT1 activity and miR-342-3p expression. Furthermore, the loss of CCSLC self-renewal exerted by miR-342-3p mimic was inverted by the overexpression of DNMT1 or FoxM1. Furthermore, DNMT1 and FoxM1 were recognized as straight targets by miR-342-3p in HeLa-derived CCSLCs.

CONCLUSION

Our findings suggested that a novel DNMT1/miR-342-3p/FoxM1 signal axis promotes CCSLC self-renewal and presented a potential target for the treatment of CC through suppression of CCSLC self-renewal. However, this pathway has been previously implicated in CC, as evidenced by prior studies showing miR-342-3p-mediated downregulation of FoxM1 in cervical cancer cells. Additionally, research on liver cancer further supports the involvement of miR-342-3p in suppressing FoxM1 expression. While our study contributed to this body of knowledge, we did not present a completely novel axis but reinforced the therapeutic potential of targeting the DNMT1/miR-342-3p/FoxM1 axis to suppress CCSLC self-renewal in CC treatment.

Keywords: DNA methyltransferase 1; Cancer stem cell; Cervical cancer; MiR-342-3p; Forkhead box M1

Core Tip: The study revealed a novel DNA methyltransferase 1 (DNMT1)/miR-342-3p/Forkhead box M1 (FoxM1) signaling axis that enhances self-renewal of cervical cancer stem cell-like cells (CCSLCs). Knockdown of DNMT1 or miR-342-3p elevation inhibits CCSLC self-renewal, while FoxM1 suppression also attenuates this process. Overexpression of DNMT1 or FoxM1 reverses the effects of miR-342-3p mimic. This signaling pathway presents a potential therapeutic target for inhibiting CCSLC self-renewal in cervical cancer, offering new insights for targeted treatment strategies.