Shu Chih-Wen, Bee Youn-Shen, Chen Jiunn-Liang, Tsen Chui-Lien, Tsai Wei-Lun, Sheu Shwu-Jiuan
Institute of Biopharmaceutical Sciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan.
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan.
Diagnostics (Basel). 2021 Feb 12;11(2):296. doi: 10.3390/diagnostics11020296.
To investigate the association of autophagy-related gene expression with age-related macular degeneration (AMD).
Patients with AMD were recruited for analysis by conjunctival impression cytology. mRNA was assessed by real-time polymerase chain reaction (RT-PCR) to evaluate whether the expression of 26 autophagy-related genes (ATGs) was correlated with AMD. Further studies on cell viability and autophagic flux in response to oxidative stress by H2O2 were performed in human retinal pigment epithelial (RPE) cell lines based on the results of impression cytology.
Both the neovascular AMD (nAMD) and polypoidal choroidal vasculopathy (PCV) groups had significantly higher mRNA levels of gamma-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) than the control group, but there was no significant difference between these two groups. Age difference existed only in the AMD group. GABARAPL1 and MAP1LC3B mRNA expression increased significantly after acute oxidative stress in adult retinal pigment epithelial (ARPE-19) cells. Cell viability significantly increased and decreased in the cells harboring GABARAPL1 expression vector and silenced with siRNA against GABARAPL1, respectively, during short-term oxidative stress, whereas viability increased in the GABARAPL1-silenced cells after long-term oxidative stress. Silencing GABARAPL1 itself caused a reduction in autophagic flux under both short and long-term oxidative stress.
Our study showed the possibility of assessing autophagy-related gene expression by conjunctival impression cytology. GABARAPL1 was significantly higher in AMD. Although an in vitro study showed an initial protective effect of autophagy, a cell viability study revealed the possibility of a harmful effect after long-term oxidative injury. The underlying mechanism or critical factors require further investigation.
研究自噬相关基因表达与年龄相关性黄斑变性（AMD）的关联。
招募AMD患者，通过结膜印片细胞学进行分析。采用实时聚合酶链反应（RT-PCR）评估mRNA，以评价26个自噬相关基因（ATG）的表达是否与AMD相关。基于印片细胞学结果，在人视网膜色素上皮（RPE）细胞系中进一步研究过氧化氢（H2O2）诱导的氧化应激对细胞活力和自噬通量的影响。
新生血管性AMD（nAMD）组和息肉状脉络膜血管病变（PCV）组的γ-氨基丁酸受体相关蛋白样1（GABARAPL1）和微管相关蛋白1A/1B轻链3B（MAP1LC3B）mRNA水平均显著高于对照组，但两组之间无显著差异。年龄差异仅存在于AMD组中。在成人视网膜色素上皮（ARPE-19）细胞中，急性氧化应激后GABARAPL1和MAP1LC3B mRNA表达显著增加。在短期氧化应激期间，分别携带GABARAPL1表达载体和用针对GABARAPL1的小干扰RNA（siRNA）沉默的细胞中，细胞活力显著增加和降低，而在长期氧化应激后，GABARAPL1沉默的细胞中活力增加。在短期和长期氧化应激下，沉默GABARAPL1本身均导致自噬通量降低。
我们的研究表明通过结膜印片细胞学评估自噬相关基因表达的可能性。AMD中GABARAPL1显著升高。虽然体外研究显示自噬具有初始保护作用，但细胞活力研究揭示了长期氧化损伤后可能产生有害作用。其潜在机制或关键因素需要进一步研究。
