第7/9型離胺酸甲基轉移酶在Sirtuin 1蛋白質表現及細胞衰老之角色探討

SET7/9 lysine methyltransferase modulates Sirtuin 1 protein expression and cellular senescence in renal tubular cells

顏夷岑(Yi-Tsen Yen)

指導教授 ： 林庭慧

中山醫學大學/醫學科技學院/生物醫學科學學系/碩士(2021年)

https://doi.org/10.6834/csmu202100256

摘要

硫酸吲哚酚（IS）是一種典型的尿毒症毒素，由於其腎毒性，在慢性腎臟疾病（CKD）的發展中非常重要。 SET7/9是一種蛋白質離氨酸甲基轉移酶，可催化組蛋白H3的離氨酸甲基化，影響染色質結構並參與基因調控。我們的研究目標，旨在闡明SET7/9 在 IS 調節的抗老化蛋白質表達和細胞衰老過程中是否扮演調控角色。我們使用1200 μM IS處理NRK-52E細胞24小時，並藉由Western Blot分析檢測抗老化蛋白質（包括Klotho、sirtuin1）的蛋白質表達。同時，也檢視了細胞衰老標記，包括衰老相關的 β-半乳糖苷酶 (SA-β-gal) 染色、Cdk 抑製劑蛋白質 p21 和 p16 的表達增加以及 p53 轉錄因子的活化，也利用免疫熒光顯微鏡檢視了 IS 對 p16 蛋白質螢光信號和 NPM1 蛋白質在核中位置的影響。我們的結果表明，IS 減弱了抗老化蛋白質（Klotho 和 Sirtuin1）的表達，並通過在 NRE-52 E 細胞中表達 β-半乳糖苷酶、p16、p21和p53來誘導細胞衰老。 NRK-53E細胞使用 SET7/9 抑製劑或通過 siRNA 沉默 SET7/9 ，受IS 抑制的 Sirtuin1 蛋白質表現量恢復。此外，當 SET 7/9 被抑制時，IS 處理的 NRK-2E 細胞中細胞衰老標誌物 β-半乳糖苷酶、p21、p16、p53 的上調減弱。這些實驗結果說明，SET7/9蛋白質在調控抗老化蛋白質 Sirtuin1表達及細胞衰老上扮演重要角色。

關鍵字

硫酸吲哚酚 ； 細胞衰老 ； 核仁壓力 ； SIRT1 ； 第7/9型離胺酸甲基轉移酶

並列摘要

Indoxyl sulfate (IS), a typical uremic toxin, is involved in the development of chronic kidney disease (CKD) with its nephrotoxicity. SET7/9 is a protein lysine methyltransferase which catalyze lysine methylation of histone H3, affecting chromatin structure and participates in gene regulation. Our study aimed to clarify the regulatory role of SET7/9 in IS-modulated anti-aging protein expression and cell senescence. Kidney epithelial cells (NRK-52E cells) were treated with 1200 μM IS for 24 hours, and protein expression of anti-aging proteins, including Klotho, sirtuin1 was detected by Western Blot analysis. Biomarker of senescent cells including senescence-associated β-galactosidase (SA-β-gal) staining, increased expression of the Cdk inhibitor proteins p21 and p16, and activation of p53 transcription factor were examined in IS-treated NRK-52E cells. The effect of IS on P16 protein fluorescent signal and NPM1 protein subnuclear localization were explored by immunofluorescence microscopy. Our results demonstrated that IS attenuated anti-aging proteins (Klotho and sirtuin1) expression and induced cell senescence with expression of β-galactosidase, p16, p21 and p53 in NRE-52 E cells. Treatment of NRK-53E cells with either SET7/9 inhibitor or silencing of SET7/9 by siRNA restored IS- suppressed sirtuin1 protein expression. Moreover, upregulation of cell senescence marker β-galactosidase, p21, p16 and p53 in IS-treated NRK-2E cells was attenuated when SET7/9 was inhibited. These results indicated that SET7/9 protein plays an important role in modulating IS-induced cell senescence and anti-aging sirtuin1 proteins expression.

並列關鍵字

Indoxyl sulfate ； Cell senescence ； nucleolus stress ； SIRT1 ； SET7/9

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