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Abstract
High-mobility group A1 (HMGA1) is a non-histone chromosomal protein, which is known as ‘architectural’ transcription factor that facilitates the assembly of ‘enhanceosome.’ Because of its elevated expression in a number of human malignancies, with barely minimal levels in healthy adults, HMGA1 is considered as potential ‘tumor marker.’ Therefore, we looked at the inhibition of hmga1 using anti-gene strategy, as an attractive therapeutic approach. This was achieved by two triplex forming oligonucleotides (TFOs), TFO1 and TFO2 targeted to the promoter of hmga1 at positions, −284–−304 and −2800–−2826, respectively. The stability of two DNA triplexes was characterized using a variety of biophysical and thermodynamics techniques and was confirmed by gel retardation assay using γ-32P [ATP]. The efficacy of TFOs on HMGA1 expression was evaluated in HeLa cells using MTT assay, Flow cytometry, Western blot, and RT-PCR. Results revealed that DNA Triplex1 formed by TFO1 is more stable and stronger than the corresponding Triplex2. Although both TFOs downregulated hmga1 expression at mRNA and protein levels and caused apoptotic cell death in HeLa cell line, TFO1 demonstrated a greater effect at low concentration which corroborates well with the stability data. Thus, TFO-mediated inhibition of hmga1 expression can be a promising strategy for the development of novel anti-cancer therapeutics.
Acknowledgments
We wish to thank Prof. Kalpana Luthra, Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India, for cell culture work. Md. Zahid Akhter is grateful to Indian Council of Medical Research (ICMR) for the award of Senior Research Fellowship (3/1/3/JRF-2008/ MPD).
Funding
This work was funded by Department of Science and Technology, Government of India [grant number DST No: SR/SO/BB-23/2010].