Korean J Physiol Pharmacol.  2021 Jan;25(1):51-58. 10.4196/kjpp.2021.25.1.51.

Oxidative stress-induced aberrant G9a activation disturbs RE-1-containing neuron-specific genes expression, leading to degeneration in human SH-SY5Y neuroblastoma cells

Affiliations
  • 1Department of Biology, College of Natural Science, Chosun University, Korea
  • 2Department of Integrative Biological Science, BK21 FOUR Education Research Group for Age-Associated Disorder Control Technology, Chosun University, Korea
  • 3Department of Cellular and Molecular Medicine, College of Medicine, Chosun University, Gwangju 61452, Korea
  • 4Department of Bio Research and Business Development, Biot Korea Inc., Gwangju 61001, Korea
  • 5Department of Otolaryngology, Chonnam National University Medical School, Gwangju 61469, Korea

Abstract

Oxidative stress-induced neurodegeneration is one of several etiologies underlying neurodegenerative disease. In the present study, we investigated the functional role of histone methyltransferase G9a in oxidative stress-induced degeneration in human SH-SY5Y neuroblastoma cells. Cell viability significantly decreased on H2O2treatment; however, treatment with the G9a inhibitor BIX01294 partially attenuated this effect. The expression of neuron-specific genes also decreased in H2O2 -treated cells; however, it recovered on G9a inhibition. H2O2 -treated cells showed high levels of H3K9me2 (histone H3 demethylated at the lysine 9 residue), which is produced by G9a activation; BIX01294 treatment reduced aberrant activation of G9a.H3K9me2 occupancy of the RE-1 site in neuron-specific genes was significantly increased in H2O2 -treated cells, whereas it was decreased in BIX01294-treated cells. The differentiation of H2O2 -treated cells also recovered on G9a inhibition by BIX01294. Consistent results were observed when used another G9a inhibitor UCN0321. These results demonstrate that oxidative stress induces aberrant activation of G9a, which disturbs the expression of neuron-specific genes and progressively mediates neuronal cell death. Moreover, a G9a inhibitor can lessen aberrant G9a activity and prevent neuronal damage. G9a inhibition may therefore contribute to the prevention of oxidative stress-induced neurodegeneration.

Keyword

Epigenetics; Histone methyltransferase; Neurodegenerative disease; Oxidative stress; Transcription factors

Figure

  • Fig. 1 Protective effects of BIX01294 in oxidative stress-damaged SH-SY5Y cells. (A) Toxicity was measured by the MTT assay in SH-SY5Y cells that were treated with 0–2 μM BIX01294 (t-test, *p < 0.05, mean ± SD, n = 3). (B) SH-SY5Y cells were treated with 0–100 μM H2O2 for 12 h (t-test, *p < 0.05, **p < 0.005, mean ± SD, n = 3). (C) SH-SY5Y cells were incubated with 0–1.5 μM BIX01294, then treated with or without 100 μM H2O2 (t-test, ##p, **p < 0.005, mean ± SD, n = 3). (D) SH-SY5Y cells were treated with 1 μM BIX01294 for 0–24 h, then treated with or without 100 μM H2O2 for 12 h (t-test, #p < 0.05, **p < 0.005, mean ± SD, n = 3). (E, F) Immunoblot analysis was performed with antibodies specific to cleaved caspase-3 in H2O2-treated SH-SY5Y cells, with or without BIX01294 treatment (t-test, *p < 0.05, mean ± SD, n = 3). Ctrl indicates control cells, which were not treated with BIX01294 or H2O2. BIX indicates BIX01294.

  • Fig. 2 Oxidative stress-induced reduction in the expression of neuron-specific genes was partially restored by G9a inhibition. (A) The expression levels of neuron-specific genes in H2O2-treated SH-SY5Y cells (H2O2) and in SH-SY5Y cells treated with both H2O2 and BIX01294 (BIX-H2O2) were measured by real-time PCR; these expression levels were compared with those in SH-SY5Y cells in normal conditions (Ctrl; t-test, #p, *p < 0.05, ##p, **p < 0.005, mean ± SD, n = 4). *p, **p (vs. control), #p, ##p (vs. 100 μM H2O2-treated cells). (B) The neuron-specific proteins Nestin, NF-M, and MAP-2 were examined by immunoblot analysis in H2O2-treated SH-SY5Y cells with or without BIX01294 treatment. (C) Protein expression levels were quantified using ImageJ software. β-actin was used as the internal standard.

  • Fig. 3 G9a is aberrantly activated by oxidative stress and disrupts the expression of RE-1-containing neuron-specific genes. (A) G9a activity was measured by immunoblot analysis with an antibody specific for H3K9me2 (top panel). The ratio of H3K9me2:H3 expression was measured using ImageJ software (bottom panel) (t-test, *p < 0.05, mean ± SD, n = 4; vs. control). (B) For the ChIP assay, SH-SY5Y cells treated with H2O2, with or without BIX01294 treatment (indicated by H2O2 or BIX-H2O2), were immunoprecipitated with H3K9me2 antibody, then analyzed by real-time PCR using primers for the RE-1 sites of NF-M, SYN1, SNAP25, and MAP-2 genes. *p, **p (vs. control), #p, ##p (vs. 100 μM H2O2-treated cells) (t-test, *p < 0.05, mean ± SD, n = 3).

  • Fig. 4 Neuronal differentiation in 2O2-treated SH-SY5Y cells recovered on inhibition of G9a. (A) SH-SY5Y cells were differentiated into neuron-like cells for 0–7 days. Each cell was visualized by light microscopy. (B, C) The expression levels of RE-1-containing neuron-specific proteins were examined using antibodies specific to NF-M and MAP-2 in H2O2-treated differentiated SH-SY5Y cells, with or without BIX01294 treatment. Ctrl indicates controls, which were non-differentiated SH-SY5Y cells. Sham indicates incubation with FBS 0.1% medium. β-actin was used as the internal standard.

  • Fig. 5 Recovery by another G9a inhibitor UCN0321 in H2O2-treated SH-SY5Y cells. (A) SH-SY5Y cells were differentiated into neuronal cells for 3 days. The expression levels of NF-M and MAP-2 were measured in H2O2-treated cells, with or without UCN0321 treatment. GAPDH was used as the internal standard. (B) The expression levels were quantified using ImageJ software.


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