July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Mitochondrial-targeted OGG1 Involved in the Ability to Protect 661W Cells against Oxidative Damage Caused by Tert-butyl Hydroperoxide
Author Affiliations & Notes
  • Wei Ma
    Zhongshan Ophthalmic Center, Guangzhou, China
  • Footnotes
    Commercial Relationships   Wei Ma, None
  • Footnotes
    Support  NSFC 81500740
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 1677. doi:
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    • Get Citation

      Wei Ma; Mitochondrial-targeted OGG1 Involved in the Ability to Protect 661W Cells against Oxidative Damage Caused by Tert-butyl Hydroperoxide. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1677.

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Abstract

Purpose : The aim of this study was to investigate the effect of mito-OGG1 overexpression or OGG1 knockdown on oxidative damage induced by tert-butyl hydroperoxide (TBH) in 661W cells.

Methods : Lentivirus-mediated mito-OGG1 overexpression or OGG1 knockdown of 661W cell stably transfected strains was established, with Real-time Quantitative PCR (QPCR) and Western blot (WB) employed to assess OGG1 expression levels. COXIV-OGG1 co-localization, cell identification, nitrotyrosine and 8-OHdG were observed by immunofluorescence. TBH cytotoxicity test on 661W cells was measured with the CCK-8 assay. Mitochondrial ROS production was detected with MitoSOX live cell fluorescent probes. DNA fragmentation was detected by TUNEL staining, and early and late apoptosis was detected by flow cytometry after PE/7-AAD staining. WB was used to detect the levels of caspase 3, Cleaved-Caspase 3, Bcl-2, Bax and MnSOD. The PCR product was run on agarose gel electrophoresis to observe the damage of mtDNA, and the comet assay was used to detect the level of nuclear DNA damage. MDA, CAT and SOD contents are measured using the corresponding biochemical kits.

Results : The results of QPCR and western blot showed that OGG1 was over-expressed in 661W cells after transfection with plenti-OGG1-GFP, while the expression of OGG1 in 661W cells was inhibited after pLKD-shRNA transfection. The survival rate of 661W cells decreased with increasing concentration of TBH mainly in a concentration-dependent manner, and cultured cells were identified as 661W photoreceptor cells. Overexpression of mito-OGG1 reduced the production of ROS and oxidative damage induced by TBH, reduced the rate of apoptosis in 661W cells, and OGG1 knockdown increased cell apoptosis. TBH treatment enhanced CAT activity, but OGG1 expression had no significant effect on CAT activity, MnSOD activity, and MnSOD protein expression.

Conclusions : Overexpression of mito-OGG1 protects against oxidative stress induced by TBH in 661W cell, but OGG1 knockdown may aggravate oxidative damage induced by TBH to some extent.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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