DNA methyltransferase 1/miR-342-3p/Forkhead box M1 signaling axis promotes self-renewal in cervical cancer stem-like cells and nude mice models.

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 mRNA, miR-342-3p, and FoxM1 protein were examined by quantitative real-time PCR and western blotting. carcinogenicity was assessed using a mouse xenograft model. The functional effects of the DNMT1/miR-342-3p/FoxM1 axis were examined by and 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 as well as a stronger tumorigenic potential compared to their parental cells. Moreover, quantitative PCR showed that the level was downregulated in HeLa-derived CCSLCs compared to HeLa cells. Its mimic significantly decreased and mRNA expression levels in CCSLCs. Knockdown of or mimic transfection suppressed expression, increased 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.