The DNMT1/miR-34a/FOXM1 Axis Contributes to Stemness of Liver Cancer Cells

Xiaocheng Cao; Lihua Liu; Xiaozheng Cao; Yinghong Cui; Chang Zou; A Chen; Yebei Qiu; Meifang Quan; Kaiqun Ren; Xiangding Chen; Jianguo Cao

doi:10.1155/2020/8978930

The DNMT1/miR-34a/FOXM1 Axis Contributes to Stemness of Liver Cancer Cells

J Oncol. 2020 Apr 5:2020:8978930. doi: 10.1155/2020/8978930. eCollection 2020.

Authors

Xiaocheng Cao^{1

2

3}, Lihua Liu^{4

5}, Xiaozheng Cao^{4

5}, Yinghong Cui^{1

2}, Chang Zou^{5

6}, A Chen^{1

2}, Yebei Qiu^{1

2}, Meifang Quan^{1

2}, Kaiqun Ren^{1

2}, Xiangding Chen³, Jianguo Cao^{1

2}

Affiliations

¹ Key Laboratory of Study and Discover of Small Targeted Molecules of Hunan Province, Medical College, Hunan Normal University, Changsha, Hunan 410013, China.
² Department of Pharmaceutical Science, Medical College, Hunan Normal University, Changsha, Hunan 410013, China.
³ Laboratory of Molecular and Statistical Genetics, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
⁴ Pharmacy Department, The Second Clinical Medical School of Jinan University, Shenzhen People's Hospital, Shenzhen 518020, China.
⁵ Shenzhen Public Service Platform on Tumor Precision Medicine and Molecular Diagnosis, Shenzhen People's Hospital, Shenzhen 518020, China.
⁶ Clinical Medical Research Center, The Second Clinical Medical School of Jinan University, Shenzhen People's Hospital, Shenzhen 518020, China.

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.