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

Kim, Ho-Tae; Ohn, Takbum; Jeong, Sin-Gu; Song, Anji; Jang, Chul Ho; Cho, Gwang-Won

doi: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 원문보기

The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology, v.25 no.1, 2021년, pp.51 - 58

Kim, Ho-Tae (Department of Biology, College of Natural Science, Chosun University) , Ohn, Takbum (Department of Cellular and Molecular Medicine, College of Medicine, Chosun University) , Jeong, Sin-Gu (Department of Biology, College of Natural Science, Chosun University) , Song, Anji (Department of Biology, College of Natural Science, Chosun University) , Jang, Chul Ho (Department of Otolaryngology, Chonnam National University Medical School) , Cho, Gwang-Won (Department of Biology, College of Natural Science, Chosun University)

Abstract ▼ AI-Helper

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 H2O2 treatment; 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.

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가설 설정

In this study, we examined the functional role of G9a in oxidative stress-induced neurodegeneration. We found that aberrant activation of G9a causes epigenetic disruption of the expression of neuron-specific genes in human neuroblastoma cells exposed to H₂O₂ (12 h), thereby leading to neurodegeneration.

제안 방법

To investigate the mechanism by which H₂O₂-damaged cells recover on inhibition of G9a, SH-SY5Y cells were incubated with or without BIX01294, prior to exposure to H₂O₂. The cells were then differentiated in neuronal induction medium.

대상 데이터

Control SH-SY5Y cells were incubated in 10% FBS medium for 3 days. Images were captured with a digital camera (i-Solution IMTcam3; Canon, Tokyo, Japan) under a microscope (Nikon ECLIPSE TS100; Nikon, Tokyo, Japan).
The SH-SY5Y human neuroblastoma cell line was purchased from KCLB (Korean Cell Line Bank, Seoul, Korea). SH-SY5Y cells were cultured in a 100-mm dish (SPL, Pocheon, Korea) in accordance with the manufacturer’s recommendations.
The real-time PCR cycling parameters were as follows: 95℃ for 10 min, followed by 40 cycles of 15 sec at 95℃ and 1 min at 60℃. The primers used in this study were synthesized by Ge- noTech (GenoTech Corp., Daejeon, Korea) and IDT (Integrated DNA Technologies Inc., Coral.,ille, IA, USA). The primers are summarized in Supplementary Table 1.

이론/모형

Cell viability of BIX01294-treated cells was evaluated using the MTT assay (Sigma-Aldrich, St. Louis, MO, USA), in accordance with the manufacturer’s instructions. Briefly, 2 × 10³ SH-SY5Y neuroblastoma cells were seeded onto 96-well plates.

성능/효과

To further examine these effects in H₂O₂- and BIX01294-treated cells, chromatin immunoprecipitation (ChIP) assays for H₃K9me2 were performed with RE-1 site-containing neuron-specific genes. H₃K9me2 occupancy of the RE-1 site of the neuron-specific genes NF-M, Syn1, SNAP-25, and Map-2 was significantly increased on exposure of cells to H₂O₂, whereas it was significantly decreased on treatment with BIX01294 (significant decrease for NF-M, SNAP-25, and Map-2; no significant difference for Syn1; Fig. 3B). These results indicate that oxidative stress induces aberrant activation of G9a, thereby disrupting neuron-specific gene expression.

후속연구

However, there were some limitations like, this study was limited to in vitro analysis, need to test with inhibition of G9a gene, and thus may not be entirely reproducible in vivo. Therefore, further studies are needed to confirm these findings regarding the relationships among aberrant activation of G9a, oxidative stress, and neurodegenerative diseases.

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