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Abstract #59295 Published in IGR 16-3
SIRT1 promote GTM cell DSBs repair and resist cellular senescence
Ren PL; Fan XJ; Yang XL; Liu MJ; Liu J; Huang JJSichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 2014; 45: 572-577
OBJECTIVE: To study the relationship between SIRT1 and glaucoma trabecular meshwork cell (GTM) cell on DNA double-strand breaks (DSBs) repair capability and resist cellular senescence. METHODS: The expressions of SIRT1 in GTM and normal trabecular meshwork (HTM) cell detected by RT-RCR and Western blot; HTM and GTM cells divided into four groups separately: Res group (treat cells with 0.5 micromol/L Resveratrol for 24 h), SIRT1-ShRNA group (cells infected with recombinant SIRT1-ShRNA), microRNA34a group (cells infected with recombinant microRNA34a) and control group. The expression level of SIRT1 in groups was detected by Western blot. SA-beta-Gal staining was applied to each group of cells at 10 h, 32 h, 3 d and 6 d to evaluate the senescence of the cells. DSBs and the expression of gamma-H2AX after treated with 1.33 mol/L H2O2 at 0 h, 1 h, 2 h were detected by comet electrophoresis and Western blot. RESULTS: The expression of SIRT1 were observed in both HTM and GTM cells, but the expression level in HTM was higher than that of GTM cells have the ability to express SIRT1, however the expression of SIRT1 was lower than HTM. Expression levels of SIRT1 presented following treads: Res > Control > microRNA34a > SIRT1-ShRNA. The dgree of senescence in GTM was higher than that in HTM cells when detected at the same time point with SA-beta-Gal staining. In the same cell line, the signs of senescence were appeared firstly and seriously in the cells treated with SIRT1-ShRNA in a time-dependent manner. Differently, after 24 h treatment with Res, the degree of senescence was decreased. The DSBs in GTM group was more than that of HTM group after treatment with oxidant when detected with Comet Electrophoresis and the the trends of the change was SIRT1-ShRNA > microRNA34a > Control > Res. The similar results also observed in the expression of gamma-H2AX. CONCLUSION: SIRT1 may be useful in predicting the development and prognosis of glaucoma; Res promotes the expression of SIRT1 significantly, and the SIRT1 may protects GTM from oxidative stress-induced DSBs, aging even apoptosis, and promotes cell cycle arrest, which may provide a new target to treat glaucoma.
Classification:
2.5.1 Trabecular meshwork (Part of: 2 Anatomical structures in glaucoma > 2.5 Meshwork)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
3.6 Cellular biology (Part of: 3 Laboratory methods)