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Hypoxia-Inducible Factor-1α and its Role in the Proliferation of Retinoblastoma Cells

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Pathology & Oncology Research

Abstract

In order to better understand the role of HIF-1α in the proliferation of the retinoblastoma cells, a siRNA knockdown of HIF-1α followed by a proliferation assay was performed. Further sequencing was then carried out in order to assess knockdown efficiency and expression of HIF-1α. Upregulation of HIF-1α gene expression in CoCl2-treated retinoblastoma cells was demonstrated via melting curve analysis from PCR tests and was further analyzed using western blot and densitometry analysis. Reduction of HIF-1α expression in retinoblastoma, post HIF-1α knockdown, was observed after siRNA transfection into Y-79 cells. Knockdown of HIF-1α resulted in a significant decrease in proliferation thereby demonstrating that HIF-1α is involved in promoting survival and proliferation in retinoblastoma cells. Stabilization of HIF-1α in retinoblastoma cells using CoCl2 was unsuccessful.

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Notes

  1. The MTT Assay

    The tetrazolium salt (MTT) is reduced in metabolically active cells by a mitochondrial dehydrogenase to form insoluble purple formazan crystals, which are solubilized by the addition of an acidified isopropanol solution. The color can then be quantified by a spectrophotometer. Because tetrazolium salts are reduced only by metabolically active cells, this assay exclusively detects viable cells. Because proliferating cell are more metabolically active, absorbance values that are higher than the control cells indicate an increase in the rate of cell proliferation. Conversely, a lower absorbance value indicates a decrease in cell proliferation.

  2. Relative and Comparative CT Methods

    Relative quantitation (ΔCT) compares transcript abundance across multiple samples, using a co-amplified internal control (in our case, GAPDH) for sample normalization. Comparative quantitation (ΔΔCT) compares the CT values of the samples of interest (in our case, the knockdowns) with a control. The CT value is the cycle number at which fluorescence crosses the threshold (i.e. exceeds background level).

  3. Densitometry

    Densitometry analysis was performed using ImageJ, a java based imaging program developed at the National Institute of Health (http://rsbweb.nih.gov/ij/).

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Competing Interests

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Ophthalmology and Pathology, The McGill University Health Center & Henry C. Witelson Ocular Pathology Laboratory, 3775 University St., Room 216, Montreal, QC, Canada, H3A 2B4

    Bruno F. Fernandes, James Coates, Carlos Quezada, Aimee Huynh, Patricia R. Odashiro & Miguel N. Burnier Jr.

  2. Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada

    Alexandre N. Odashiro

  3. Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil

    Macanori Odashiro

Authors
  1. Bruno F. Fernandes
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  2. James Coates
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  3. Alexandre N. Odashiro
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  4. Carlos Quezada
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  5. Aimee Huynh
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  6. Patricia R. Odashiro
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  8. Miguel N. Burnier Jr.
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Correspondence to Carlos Quezada.

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Fernandes, B.F., Coates, J., Odashiro, A.N. et al. Hypoxia-Inducible Factor-1α and its Role in the Proliferation of Retinoblastoma Cells. Pathol. Oncol. Res. 20, 557–563 (2014). https://doi.org/10.1007/s12253-013-9728-8

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  • DOI: https://doi.org/10.1007/s12253-013-9728-8

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