Research Article
Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance

https://doi.org/10.1016/j.yexcr.2014.05.022Get rights and content

Highlights

  • Knockdown of miR-210 decreases hypoxic GSCs stemness.

  • MiR-210 knockdown rescues MNT expression resulting in repression of Myc function.

  • Knockdown of miR-210 radiosensitized hypoxic GSCs.

  • MiR-210 might be a therapeutic target to eliminate GSCs in hypoxic niches.

Abstract

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches.

Introduction

Glioma is one of the most common and deadly adult brain tumors. Despite the combination treatment of surgical resection, radiotherapy, and chemotherapy, mean survival time of glioma patients remains about 15 months after diagnosis and only a few survive for more than 5 years [1]. In recent years, many studies have demonstrated that the fatal nature of glioma is caused by a small proportion of glioma cells, known as glioma stem cells (GSCs), which exhibit stem cell properties and show the ability of self-renewal and multi-potential differentiation, and have the ability to initiate and propagate tumors [2], [3]. Rigorous functional studies confirmed the existence of GSCs and provided evidence that GSCs promoted therapeutic resistance and relapse of glioma [4], [5], [6], [7].

GSCs are enriched in specific niches around tumor vessels and areas of necrosis [8], [9], [10], the latter associated with hypoxia. Glioma, like many other human solid tumors, usually develops local hypoxia, which promotes glioma progression by facilitating angiogenesis and metabolic adaptation and renders them resilient to radiotherapy [10]. Hypoxia has critical effects on GSCs function [11]. Hypoxia promotes the neurosphere formation of GSCs and induces the expression of stemness markers such as c-Myc, Sox2 and Oct4 [12], [13]. Cellular responses to hypoxia are commonly regulated by the hypoxia inducible factor (HIF) family of transcriptional factors. HIFs function as heterodimers consisting of an oxygen sensitive HIFα subunit and a constitutively expressed HIFβ subunit. Two HIFα proteins, HIF1α and HIF2α, are involved in GSCs maintenance and are required for GSC growth and survival [14], [15].

MiRNAs are a class of small (21–22 nucleotide in length) single-stranded noncoding RNAs, processed from much longer primary transcripts, which participate in crucial biological processes, including cell proliferation, differentiation, apoptosis, metabolism and tumorigenesis through inhibition of RNA translation or degradation of target messenger RNA (mRNA) by binding to imperfect complementary sites within the target genes’ 3’ untranslated region (UTR) [16]. Recently, it has been demonstrated that a specific set of miRNAs molecules are upregulated by hypoxia [17]. Among these hypoxia-induced miRNAs, miR-210 is a master and a unique one in its wide distribution, HIF dependence and robust upregulation in response to hypoxia [18], [19]. MiR-210 promotes cell proliferation, represses mitochondrial metabolism and induces angiogenesis by targeting several genes such as Myc antagonist (MNT), iron–sulfur cluster scaffold protein (ISCU) and ephrin-A3 (EFNA3) [20], [21], [22]. Moreover, miR-210 improves the survival of mesenchymal stem cells under anoxia and increased proliferation and stemness gene expression of adipose-derived stem cells, such as Oct4 and Rex1 [23], [24]. Recently, several groups have demonstrated that miR-210 might be a potential therapeutic target and a marker of radioresistance in tumor cells [25], [26], [27], [28]. However, whether miR-210 could serve as an excellent candidate for therapeutic intervention to hypoxic cancer stem cells remains obscure.

In this study, GSCs with stable integration of the anti-sense miR-210 were generated through lentiviral-mediated gene transfer, which followed by exposure to hypoxia, GSCs stemness, differentiation, cell cycle, apoptosis and radiosensitivity were detected to explore the effect of miR-210 downregulation on stemness and radioresistance of hypoxic GSCs and its mechanism.

Section snippets

Cell culture

The human glioma cell lines U87 and SHG44 were purchased from the Type Culture Collection of the Chinese Academy of Sciences and cultured in DMEM/F12 medium (1:1, Hyclone) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) in a humidified atmosphere containing 5% CO2 at 37 °C. GSCs (U87s and SHG44s) were enriched using a serum-free clone formation method with serum-free DMEM/F12 supplemented with 2% B27 Neuro Mix (invitrogen), 20 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic

Identification of GSCs from U87 and SHG44 cells

Both U87s cells and SHG44s cells showed strong expression of glioma stem cell markers CD133 and nestin, which were involved in self-renewal and proliferation of stem cells (Fig. 1a). To determine the percentage of cells with the unique characteristics of stem cells, quantitative analysis of CD133 and nestin positive cells were performed by flow cytometry. The results showed that U87s cells contained 88.8% CD133 positive cells and 95.7% nestin positive cells, and SHG44s cells contained 84.9%

Discussion

Hypoxia is a well recognized tumor microenvironmental factor which is associated with poor patient outcome and resistance to therapies. Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of GSCs [10]. Since GSCs are resistant to conventional therapies and responsible for recurrence, it is important to develop novel molecularly targeted therapies to target GSCs located in hypoxic niches [6], [7].

The hypoxic regulation of miR-210 was first

Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81071958) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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