Regulation of miRNA-21 by reactive oxygen species-activated ERK/NF-κB in arsenite-induced cell transformation

https://doi.org/10.1016/j.freeradbiomed.2012.02.020Get rights and content

Abstract

After acute exposure of cells to arsenic, reactive oxygen species mediate changes in cell behavior, including activation of proliferative signaling. For chronic exposure to arsenic, however, the function of reactive oxygen species in cell transformation remains poorly understood. Although microRNA-21 (miR-21) has been implicated in various aspects of carcinogenesis, its functions and molecular mechanisms in carcinogen-induced tumorigenesis are unclear. The purpose of this study was to determine if miR-21 is involved in arsenite-induced malignant transformation and to characterize the associated signaling pathways. During arsenite-induced transformation of human embryo lung fibroblast (HELF) cells, miR-21 was upregulated, and the extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) signal pathway was activated. Moreover, superoxide radical dismutase (a scavenger of superoxide) and catalase (a scavenger of hydroperoxides) blocked the arsenite-induced effects in HELF cells and mouse embryonic fibroblasts. Blockage of ERK by the inhibitor U0126 or inhibition of NF-κB p65 by siRNA or Bay 11–7082 prevented the increases in miR-21 and the decreases in Spry1, Pten, and Pdcd4, the target proteins of miR-21, induced by arsenite. As determined by a ChIP-qPCR assay, NF-κB p65 regulated miR-21 expression by binding directly to the promoter of miR-21. Further, anti-miR-21 downregulated miR-21 expression and prevented the arsenite-induced activation of ERK via the increase in Spry1, indicating that miR-21 has a feedback effect in regulating ERK activation. Overexpression of miR-21 with an miR-21 mimic and feedback activation of ERK and NF-κB via the decrease in Spry1 promoted the malignancy of HELF cells exposed to arsenite, but knockdown of miR-21 with anti-miR-21 and feedback blockage of ERK and NF-κB activation through an increase in Spry1 decreased anchorage-independent growth of arsenite-transformed cells. Thus, the transformation of HELF cells induced by chronic exposure to arsenite is mediated by increased miR-21 expression, which, in turn, is mediated by reactive oxygen species activation of the ERK/NF-κB pathway.

Graphical abstract

Highlights

► Chronic exposure to arsenite induces the transformation of HELF cells. ► Arsenite improves miR-21 expression by reactive oxygen species activation of the ERK/NF-κB pathway. ► NF-κB regulates miR-21 expression by binding directly to the promoter of miR-21. ► MiR-21 has a feedback effect in regulating ERK activation via Spry1. ► MiR-21 affects the malignancy potential of arsenite-transformed HELF cells.

Section snippets

Cell culture

Immortalized HELF cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). These cells are human sarcoma virus 40 immortalized, nontumorigenic, diploid fibroblasts from the lungs of hysterotomy-derived embryos [19]. Cells were maintained in 5% CO2 at 37 °C in Dulbecco's modified Eagle medium (DMEM; Life Technologies/Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies/Gibco), penicillin (100 U/ml),

Upregulation of miR-21 and activation of ERK and NF-κB induced by arsenite in HELF cells

To determine if arsenite increases transformation, HELF cells were incubated with or without 1.0 μM arsenite continuously. After 30 passages (about 15 weeks) of exposure, arsenite was removed from the cell culture medium before the initiation of experiments. Anchorage-independent growth is a characteristic gained by cancer cells conferring mobility without the consequence of apoptosis [18]. Colonies formed from arsenite-exposed cells were both larger (Supplementary Fig. S2A) and greater in number

Discussion

Recent mechanistic studies of arsenite carcinogenesis have directly or indirectly shown the involvement of altered epigenetic regulation in gene expression changes induced by arsenite exposure [34]. Arsenite is a well-established human carcinogen [35], [36]. A close association and positive correlation exists between arsenite exposure and increased incidences of various forms of cancer [7], [35]. Although various arsenite-regulated genes and processes have been described, many of the molecular

Acknowledgments

The authors thank Donald L. Hill (University of Alabama at Birmingham, USA) for editing. This work was supported by the Natural Science Foundations of China (30872146 and 81072327), the Research Fund for the Doctoral Program of Higher Education of China (20103234110005), the Key Program of the Educational Commission of Jiangsu Province of China (11KJA330002), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (2010).

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