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. 2013 Oct;184(2):855-60.
doi: 10.1016/j.jss.2013.04.061. Epub 2013 May 18.

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

Thomas A Mace  1 , Amy L Collins, Sylwia E Wojcik, Carlo M Croce, Gregory B Lesinski, Mark Bloomston
Affiliations

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

Thomas A Mace et al. J Surg Res. 2013 Oct.

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.

Keywords: HIF-1alpha; Hypoxia; Micro-RNA; Pancreatic cancer.

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Figures

Figure 1
Figure 1. Hypoxia induces MiR-21 and recruits HIF-1α to MiR-21 promoter in pancreatic cancer cell lines
A) Cells were grown in 21% (normoxia) or 1% oxygen (hypoxia) for 48 hours. MiR-21 was assayed by qRT-PCR and normalized to snoRNA U48. B) Immunoblot of HIF-1 α from lysates of cells cultured at normoxic or hypoxic conditions. C) ChIP analyses of recruitment of HIF-1α to the predicted hypoxia response element (HRE) region of miR-21 following treatment of AsPC-1 cells grown at normoxic or hypoxic conditions for 72 hours.
Figure 2
Figure 2. MiR-21 upregulation by hypoxia is attenuated by inhibition of HIF-1α with siRNA
BxPC-3 and AsPC-1 cells were transfected with HIF-1 α siRNA and cultured in normoxic or hypoxic conditions for 48 hours and miRNA expression was assessed by RT-PCR.
Figure 3
Figure 3. HIF-1α overexpression in normoxia
A) Western blot of HIF-1α expression 48 hours after transfection with constitutively stable pcDNA HIF-1α; B) MiR-21 overexpression through transfection of the cells with pcDNA HIF-1α by qRT-PCR in normoxia
Figure 4
Figure 4. miR-21 increases proliferation and overrides hypoxia-induced cell cycle arrest
AsPC-1 cells were transfected with mature miR-21, antisense to miR-21 and NC-non coding and were cultured for 48 hours in normoxic or hypoxic conditions. Proliferation was measured by WST-1 assay.
Figure 5
Figure 5. Pancreatic cancer cell survival is attenuated by anti-miR21 siRNA
AsPC-1 cells were treated with 100nm anti-miR21 siRNA were harvested after 48 hours of incubation and using Annexin V-FITC was stained to assess apoptosis.
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