Gastrointestinal
Hypoxia induces the overexpression of microRNA-21 in pancreatic cancer cells

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Abstract

Background

Pancreatic cancer cells exist in a hypoxic microenvironment containing numerous factors that impact tumor survival, proliferation, and metastasis. MicroRNAs (miRs) are differentially expressed in cancer but also altered by hypoxia. We hypothesized that hypoxia could induce expression of miR-21, an oncomir in pancreatic cancer cells.

Materials and methods

We examined how hypoxia regulates miR-21 expression in pancreatic cancer cell lines (BxPC-3, AsPC-1) by stem-loop RT-PCR. Chromatin immunoprecipitation assays were used to study how hypoxia alters hypoxia-inducible factor (HIF)-1α binding to the hypoxia response element of miR-21. BxPC-3 and AsPC-1 cells were transfected with a constitutively stable HIF-1α subunit or vector control (pcDNA3.1) to determine the influence of miR-21 in normoxia. The effect of mature miR-21 sense and antisense oligonucleotides on proliferation and apoptosis in hypoxic and normoxic conditions was assessed via WST-1 assay and flow cytometry.

Results

MiR-21 levels increased in all cell lines grown in hypoxic conditions versus normoxia, whereas siRNA targeting HIF-1α reduced miR-21 expression. Hypoxic conditions resulted in direct binding of HIF-1α to the predicted binding site in miR-21. Transfection with a constitutively stable HIF-1α expression plasmid in normoxia resulted in upregulated miR-21, similar to that seen in hypoxia. Cells transfected with antisense constructs targeting miR-21 had reduced proliferation and increased apoptosis in normoxia, whereas miR-21 overexpression abrogated hypoxia-associated reductions in proliferation.

Conclusions

MiR-21 is induced by hypoxia in pancreatic cancer cells via HIF-1α upregulation. MiR-21 overexpression allows cells to avoid apoptosis in a hypoxic microenvironment. Inhibition of miR-21 expression may increase cellular susceptibility to hypoxia in pancreatic cancer.

Introduction

Pancreatic cancer is the fourth most common cause of cancer-related death in the United States [1], [2]. In the best of circumstances, when disease is confined to the pancreas without lymphatic or hematogenous spread, 5-y overall survival remains less than 30% with a median survival of approximately 18–24 mo [3]. Unfortunately, the majority of patients are not amenable to resection due to locally advanced or metastatic disease. This dismal prognosis owes to the aggressive nature of the disease resulting in advanced stage at the time of presentation and its inherent resistance to chemotherapy and radiation. Much research has focused on identifying gene expression patterns that might explain its pathogenesis and aggressive nature. Thus far, results have been disappointing.

Hypoxia is an essential component of the neoplastic microenvironment, often allowing a selective advantage for tumor cells over otherwise noninvasive cells more sensitive to a low oxygen state. The evidence for hypoxia in pancreatic cancer is in their characteristic avascular appearance on computed tomography [4] and from intratumoral oxygen tension measurements [5]. Hypoxic conditions in solid malignancies may confer resistance to conventional radiation and chemotherapy [6], [7]. A functional link between hypoxia and microRNA (miR) expression was shown in colon and breast cancer cell lines by Kulshreshtha et al. [8] and in several other cancers [9], [10], [11]. Interestingly, the majority of hypoxia-associated miRs predicted in silico are differentially overexpressed in pancreatic cancer.

MicroRNA research holds the promise of providing molecular insight into carcinogenesis and disease progression. These small noncoding RNAs, when in mature form, are incorporated into the RNA-induced silencing complex, where they act by binding to a conserved sequence of the 3′ untranslated region of protein-coding target genes, leading to mRNA degradation or translational inhibition, depending on perfect or imperfect complementarity, respectively [12]. Target genes involved in apoptosis or cell cycle progression allow miRs to act as oncogenes and tumor suppressor genes [13], [14], [15]. These oncomirs have been described in both hematologic and solid tumors. One such miR, miR-21, is overexpressed in multiple malignancies including pancreatic, esophageal, lung, breast, and colon cancers [16], [17], [18], [19], [20]. This oncogenic miR has been linked to tumor aggression and carcinogenesis, in part, by preventing apoptosis [21], [22]. MiR-21 is significantly overexpressed in pancreatic cancer as detected by in situ hybridization and its strong expression predicts limited survival in patients with node-negative disease [23]. Little is known, however, of the mechanism by which upregulation of miR-21 occurs.

In this study we explored the role of hypoxia in pancreatic cancer. Of particular interest was the hypoxic regulation of miR-21 and its relevance in tumor pathogenesis. We show that miR-21 is upregulated in pancreatic cancer cells in response to hypoxia through expression of hypoxia-inducible factor (HIF)-1α, and that miR-21 expression allows for cell survival in the hypoxic environment.

Section snippets

Cell lines and culture conditions

Human pancreatic cell lines (AsPC-1 and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in 21% O2 and 5% CO2 at 37°C. Hypoxia was induced in a hypoxic chamber (Billups-Rothenberg, Inc, Del Mar, CA) with 1% oxygen in parallel with cells maintained in normoxia. O2 and CO2 were measured and controlled using dual-flow meters in order to monitor, measure, and keep

MiR-21 expression is upregulated by hypoxia in vitro

Pancreatic cancer cell lines BxPC-3 and AsPC-1 were cultured for 48 h in normoxic or hypoxic conditions and miR-21 expression was assessed by RT-PCR. Cells cultured in hypoxic conditions for 48 h resulted in significantly increased expression of HIF-1α (Fig. 1A, P = 0.0015). Given our previous report of differential miR-21 overexpression in resected human pancreatic cancers [19], we first sought to define the impact of hypoxia on miR-21 expression by qRT-PCR in human pancreatic cancer cell

Discussion

MicroRNAs are often dysregulated in tumors and have therefore been classified as oncomirs, which modulate cell proliferation and survival [26], [27]. miR-21 was among the first oncomirs identified and is overexpressed in multiple histologic subtypes of cancer, including glioblastoma, lung cancer, and leukemia [28], [29]. Increased miR-21 levels in pancreatic cyst fluid have been shown to be a predictive marker for ductal adenocarcinoma [30]. Other recent studies also provide evidence that

Acknowledgment

Research was supported by the 2010 AACR-FNAB Fellows Grant for Translational Pancreatic Cancer Research Grant Number 10-30-14-COLL (A.L.C.), NCI CA13325-01 (M.B.), and the Society of University Surgeons Junior Faculty Award (M.B.).

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    T.A.M. and A.L.C. contributed equally to this work.

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