Abstract
Background Whether DNA methyltransferase 1 (DNMT1)/miR-34a/FoxM1 signaling promotes the stemness of liver cancer stem cells (LCSCs) remains unclear. This study aimed to assess whether methylation-based silencing of miR-34a by DNMT1 contributes to stemness features via FoxM1 upregulation in LCSCs. Methods The CD133+ subgroup of MHCC97H cells sorted by MACS was used as LCSCs. DNMT1, BMI1, SOX2, and OCT4 mRNA levels, and miR-34a amounts were determined by qRT-PCR. DNMT1, CD44, and FoxM1 proteins were analyzed by immunoblot. Sphere and colony formation abilities were detected by respective assays. CD133+ cell percentages were assessed by flow cytometry. In vivo oncogenicity was evaluated using a tumor xenograft model in mice. The effects of DNMT1/miR-34a signaling on the stemness of LCSCs were examined by knockdown or overexpression of DNMT1 and/or transfection of miR-34a mimic or inhibitor using lentivirus-delivery systems. FoxM1 association with miR-34a was detected by a reporter assay. Results We here showed that LCSCs exhibited elevated DNMT1 activity and expression, lower miR-34a expression with higher promoter methylation, and stronger stemness, compared with the parental liver cancer cells. DNMT1 knockdown repressed DNMT1, increased miR-34a amounts by promoter demethylation, and reduced stemness in LCSCs, whereas DNMT1 overexpression had the opposite effects in liver cancer cells. Transfection with miR-34a mimic repressed the stemness of LCSCs, while miR-34a inhibitor significantly downregulated miR-34a and enhanced stemness, without affecting DNMT1 in liver cancer cells. MiR-34a mimic rescued the effects of DNMT1 overexpression on the stemness of LCSCs, without affecting DNMT1 expression. Finally, FOXM1 was identified as a direct target by miR-34a in LCSCs. Conclusions We revealed that aberrant activation of DNMT1 causes miR-34a promoter methylation and suppression, leading to FoxM1 upregulation by disinhibition and promotion of LCSC stemness. These findings suggest that blockage of DNMT1/miR-34a-mediated FOXM1 upregulation might suppress liver cancer by targeting LCSCs.
Highlights
Whether DNA methyltransferase 1 (DNMT1)/miR-34a/Forkhead box M1 (FoxM1) signaling promotes the stemness of liver cancer stem cells (LCSCs) remains unclear. is study aimed to assess whether methylation-based silencing of miR-34a by DNMT1 contributes to stemness features via FoxM1 upregulation in LCSCs
We initially explored whether CD133+ spheres from MHCC97H cells used as LCSCs are involved in DNMT activation and methylation silencing of miR-34a
We found that DNMT1 activation downregulated miR-34a and enhanced mir-34a promoter methylation in LCSCs (Figures 1(c) and 1(d))
Summary
Whether DNA methyltransferase 1 (DNMT1)/miR-34a/FoxM1 signaling promotes the stemness of liver cancer stem cells (LCSCs) remains unclear. is study aimed to assess whether methylation-based silencing of miR-34a by DNMT1 contributes to stemness features via FoxM1 upregulation in LCSCs. Whether DNA methyltransferase 1 (DNMT1)/miR-34a/FoxM1 signaling promotes the stemness of liver cancer stem cells (LCSCs) remains unclear. Is study aimed to assess whether methylation-based silencing of miR-34a by DNMT1 contributes to stemness features via FoxM1 upregulation in LCSCs. Methods. We here showed that LCSCs exhibited elevated DNMT1 activity and expression, lower miR-34a expression with higher promoter methylation, and stronger stemness, compared with the parental liver cancer cells. DNMT1 knockdown repressed DNMT1, increased miR-34a amounts by promoter demethylation, and reduced stemness in LCSCs, whereas DNMT1 overexpression had the opposite effects in liver cancer cells. We revealed that aberrant activation of DNMT1 causes miR-34a promoter methylation and suppression, leading to FoxM1 upregulation by disinhibition and promotion of LCSC stemness. Whether abnormal DNMT1 expression resulting in miR-34a downregulation induces and maintains LCSC features remains largely unclear
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