The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells.

Xiao Liang,Wanchun Wang,Xiang Li,Chang Xu

doi:10.1155/2019/7028901

Abstract

The DNA methyltransferase 1 (DNMT1)/miR-34a axis promoted carcinogenesis of various types of cancers. However, no literature reported its contribution to the stemness of osteosarcoma cancer stem-like cells (OSLCs). We sought to determine whether the DNMT1/miR-34a axis facilitates the stemness of OSLCs. We here revealed the higher DNMT1 activity and expression, lower miR-34a expression with high methylation of its promoter, and stronger stemness of OSLCs, as manifested by elevated sphere and colony formation capacities; CD133, CD44, ABCG2, Bmi1, Sox2, and Oct4 protein amounts in vitro; and carcinogenicity in a nude mouse xenograft model, when compared to the parental U2OS cells. 5-Azacytidine (Aza-dC) repressed DNMT1 activation and upregulated miR-34a expression by promoter demethylation and suppressed the stemness of OSLCs in a dose-dependent manner. DNMT1 knockdown increased miR-34a and reduced the stemness of OSLCs. Transfection with a miR-34a mimic repressed the stemness of OSLCs but did not alter DNMT1 activity and expression. Conversely, DNMT1 overexpression declined miR-34a levels, promoting the stemness of U2OS cells. Transfection with a miR-34a inhibitor enhanced the stemness of U2OS cells, without affecting the DNMT1 activity and expression. Importantly, reexpression of miR-34a could rescue the effects of DNMT1 overexpression on miR-34a inhibition as well as the stemness promotion without affecting the activity and expression of DNMT1. Our results revealed that aberrant activation of DNMT1 caused promoter methylation of miR-34a, leading to miR-34a underexpression, and the role of the DNMT1/miR-34a axis in promoting and sustaining the stemness of OSLCs.

Highlights

Osteosarcoma (OS) is the most common bone-derived solid cancer in children and adolescents and originates from mesenchymal cells of osteoblast origin [1, 2]
DNA methyltransferase 1 (DNMT1) Activation and miR-34a Underexpression Are Associated with a Cancer Stem-like Cell (CSLC) Feature in the U2OS Cell Line
To examine whether DNMT1 activation was required for the acquisition of a cancer stem-like cells (CSLCs) feature, we examined the inhibition of DNMT1 activity by Aza-dC on the capacities of sphere formation and clonogenicity and the amounts of CD133, CD44, ABCG2, Bmi1, Sox2, and Oct4 in osteosarcoma cancer stem-like cells (OSLCs)

Summary

Introduction

Osteosarcoma (OS) is the most common bone-derived solid cancer in children and adolescents and originates from mesenchymal cells of osteoblast origin [1, 2]. Accumulating evidence supported the notion that a small subpopulation of cells with stem-like characteristics called cancer stem-like cells (CSLCs) are the most cause for cancer metastasis and chemoresistance owing to their stronger stemness [4]. MicroRNAs (miRNAs) regulate the function and property of CSLCs, and dysregulation was involved in the stemness of prostate cancer and Ewing’s sarcoma [5, 6]. Recent studies have reported that miR-34a is underexpressed in various cancers such as Ewing’s sarcoma [8], colorectal cancer [9], and OS [10, 11]. MiR-34a regulated colon CSLCs [12] and inhibited breast cancer stemness [13]. Liu et al demonstrated that miR-34a inhibits prostate CSLCs by directly repressing CD44 [14]. The study by Zhang et al [15] revealed that

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