γ-GCS磷酸化介导微囊藻毒素-LR致肝癌发病机制的研究

Microcystin-LR (MC-LR), the most important toxin in cyanobacteria-polluted water, was reported to be responsible for the increased incidence of hepatocellular carcinoma. However, the mechanisms of its carcinogenic action are still unclear. We speculated that MC-LR may change the expression of γ-glutamylcysteine synthetase (γ-GCS) through hyperphosphorylation of γ-GCS, which maybe caused by direct protein phosphatase 2A (PP2A) inhibition. In order to test the hypotheses, MC-LR is initially chosen to establish a malignant transformation model in immortalized hepatocyte cell line. Alterations in PP2A, phosphorylation state, gene expression, enzyme activities of γ-GCS and oxidative stress status are then examined in the transformed cells and nude mice with malignant tumours. Subsequently, the expression of γ-GCS in the transformed cell is down-regulated by siRNA transfection and up-regulated by cDNA transfection, in order to determine the role of γ-GCS in MC-LR-induced carcinogenesis. Further, epidemiological investigations are performed to verify the toxicological findings among people from high- or low-incidence area of Guangxi. The aims of this study are to define the roles of γ-GCS phosphorylation and oxidative damage induced by MC-LR in hepatocarcinogenesis, and provide the evidence needed to clarify the mechanisms of carcinogenesis and early biomarkers for MC-LR exposure.

微囊藻毒素-LR（MC-LR）是水体水华产生的有毒藻类毒素，流行病学调查显示MC-LR与肝癌的高发相关，但MC-LR致癌机制并不十分清楚。MC-LR暴露可改变γ-谷氨酰半胱氨酸合成酶（γ-GCS）的表达及活性，推测可能与MC-LR抑制蛋白磷酸酶2A（PP2A）导致γ-GCS过磷酸化有关。本课题拟建立MC-LR诱导永生化正常人肝细胞的恶性转化和癌变模型，动态观察细胞中PP2A活性、γ-GCS状态（磷酸化、蛋白表达、酶活性）与氧化应激水平的变化及其关系；采用cDNA转染和siRNA正、反向干预γ-GCS基因，探讨γ-GCS在MC-LR致癌过程中的作用；并利用广西肝癌高/低发现场人群验证毒理学实验的发现，揭示γ-GCS磷酸化和氧化损伤在MC-LR致癌过程中的作用，为进一步阐明MC-LR的致癌分子机制和早期效应标志提供科学依据。

本课题通过建立微囊藻毒素-LR（MC-LR）诱导的人肝细胞WRL68的恶性转化模型，动态观察细胞中蛋白磷酸酶2A（PP2A）活性、γ-谷氨酰半胱氨酸合成酶（γ-GCS）磷酸化、蛋白表达、酶活性与氧化应激水平的变化及其相互关系；利用慢病毒载体构建γ-GCS调节亚基（GCLC）过表达的WRL68细胞株，经MC-LR诱导细胞恶性转化，研究GCLC基因过表达对MC-LR诱导肝细胞恶性转化的影响。结果发现，MC-LR低剂量长期染毒诱导WRL68细胞发生恶性转化过程中伴随PP2A酶活性持续降低，GCLC蛋白磷酸化水平持续增加，引起GCLC的mRNA表达、蛋白表达水平下调，γ-GCS酶、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性以及谷胱甘肽（GSH）含量逐渐下降，8-羟基脱氧鸟苷（8-OHdG）和丙二醛（MDA）含量逐渐升高。GCLC过表达可部分抑制WRL68细胞增殖、锚着独立性生长以及裸鼠成瘤性。MC-LR暴露和非暴露者的病例-对照研究发现，广西肝癌高发区健康人群血清MC-LR水平高于低发区，肝癌人群血清MC-LR水平高于健康人群，MC-LR高暴露的健康人群血清GSH含量低于MC-LR低暴露人群。上述结果表明γ-GCS和氧化损伤在MC-LR致癌过程中可能发挥着重要作用。本结果后续可用于MC-LR的致癌分子机制和早期效应标志的探索，为肝癌的预防和控制提供科学依据。