The DNMT1miR-34aFOXM1 Axis Contributes to Stemness of Liver Cancer Cells

الملخص EN

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.