Nrf2蛋白Neh3结构域酪氨酸磷酸化修饰位点变异影响胆囊癌细胞化疗敏感性的机制研究

As we all know that gallbladder cancer cell show strong resistence to many kinds of chemotherapy drugs, but the mechanism is still not well known. Our previous finding show that the lower ROS levels and higher expression of membrane transport protein （ABCG2 and MRP1) in gallbladder cancer cells correlate well with stronger drug resistance. Our preliminary experiment showed that Nrf2, a central regulator of intracellular redox state,can regulate ROS levels and impact on chemotherapy sensitivity of gallbladder cancer cells through influencing the expression levels of antioxidant related-genes( NQO1, HMXO1, SOD2 and GPX) and drug pump protein ABGC2.We are also the first to find that some gallbladder cancer tissuse has a point mutation (Y576F) in Nrf2 Neh3 domain, which will lead to a upregulation of downstream antioxidant genes and ABCG2, therefore contributing to a stronger drug resistence. In this study, via the cell, animal and clinical experiments, we will for the first time reveal that Nrf2 Y576f mutation in gallbladder cancer cells will later block phosphorylation leading to a high cumulative level of Nrf2 in nucleus. The high concentration of Nrf2 in nucleus will then induce higher expressions of downstream antioxidant genes, membrane transport proteins and detoxification enzymes, resulting in lowered ROS level,reduced drug concentration, and accelated metobolism rate of chemotherapy drugs respectively in gallbladder cancer cells. All these will at last contributing to a higher drug resistence. We will also investigate the Nrf2 Y576F point mutation whether can be a molecular diagnostic marker for the chemosensitivity of gallbladder cancer.

胆囊癌细胞对化疗药物不敏感，但其机制并不清楚。我们的前期研究发现，胆囊癌细胞内低ROS水平和膜转运蛋白（ABCG2及MRP1）高表达与其耐药性相关。预实验发现细胞内氧化还原状态的中枢调节者Nrf2通过调控抗氧化基因（NQO1、HMXO1、SOD2及GPX）及膜转运蛋白ABCG2的转录，进而调节细胞内ROS水平，影响胆囊癌细胞对化疗药物的敏感性。我们首次发现人体胆囊癌组织内存在Nrf2 Neh3结构域Y576F位点突变导致Nrf2下游抗氧化基因和ABCG2表达升高，耐药性增强。本研究将从临床样本、细胞和动物三个层面首次揭示：胆囊癌患者Nrf2 Y576F位点突变→Nrf2磷酸化受阻及核内累积增加→下游抗氧化基因、膜转运蛋白及解毒酶的表达增加→细胞内ROS降低、化疗药物浓度降低及药物代谢速率加快→胆囊癌细胞耐药性增强，探讨Nrf2 Y576F位点突变是否可以作为胆囊癌化疗敏感性的分子诊断标志。

胆囊癌细胞对化疗药物不敏感，然而其机制尚不清楚。我们的前期研究发现，胆囊癌细胞内低ROS 水平和膜转运蛋白（ABCG2 及MRP1）高表达与其耐药性相关。预实验发现细胞内氧化还原状态的中枢调节者Nrf2 通过调控抗氧化基因（NQO1、HMXO1、SOD2 及GPX）及膜转运蛋白ABCG2 的转录，进而调节细胞内ROS 水平，影响胆囊癌细胞对化疗药物的敏感性。我们首次发现人体胆囊癌组织内存在Nrf2 Neh3 结构域酪氨酸位点突变导致Nrf2 下游抗氧化基因和ABCG2 表达升高，耐药性增强。本研究将从临床样本、细胞和动物三个层面首次揭示：胆囊癌患者Nrf2酪氨酸位点突变→Nrf2 磷酸化受阻及核内累积增加→下游抗氧化基因、膜转运蛋白及解毒酶的表达增加→细胞内ROS 降低、化疗药物浓度降低及药物代谢速率加快→胆囊癌细胞耐药性增强，探讨Nrf2相关突变位点是否可以作为胆囊癌化疗敏感性的分子诊断标志。