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 mechanism...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 HeLaderived 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 methylationspecific 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.展开更多
基金Supported by Guangzhou Basic and Applied Basic Research Foundation,No.202201010121Medical Joint Fund of Jinan University,No.YXZY2024014 and No.YXJC2022001+2 种基金Hospital Achievement Transformation and Cultivation Project,No.ZH201911the Key Discipline Project of Guangdong Province,No.2019-GDXK-0016and the Medical Science and Technology Research Foundation of Guangdong Province,No.B2021145.
文摘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 HeLaderived 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 methylationspecific 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.
