微量元素硒调控Nrf2信号通路的机制研究

Esophageal cancer, the sixth most commonly occurring cancer and the fifth most common cause of cancer death in the world, occurs at a very high frequency in certain areas of China. The epidemiological studies performed in Linxian, China, show a significantly inverse association between serum selenium levels and the incidence of esophageal cancer. Supplementation with Selenium-containing compounds was associated with a significantly lower cancer mortality rate in a study conducted in Linxia,China. Thus, selenium could be a potential anti-esophageal cancer agent. Cancer chemopreventive agents inhibit carcinogenesis most relying on triggering off activation of Nrf2 pathway and epigenetic regulation. However,the exact machanism of selenium in the prevention of esophageal carcinogenesis is still unclear. In this study, we will explore function of methylseleninic acid(MSA) in regulating Nrf2 pathway.Also,the HDAC activity and protein degradation, the Keap1 protein degradation will be detected after MSA treatment. The accurate mechanism and the regulation network in inhibiting esophageal carcinogenesis by MSA will be investigated. Our results will demonstrate that MSA can inhibit esophageal carciogenesis and also support that selenium will potentially be used as a chemopreventive agent against ESCC.

食管癌是我国特有的高发肿瘤之一，具有明显的地域聚集性。流行病学资料表明硒缺乏与食管癌的发生风险密切相关。针对食管癌高发区进行的大规模人群营养干预研究显示，补充含硒化合物可以降低食管癌的发病率，提示硒具有化学预防的作用。化学预防药物多通过激活Nrf2通路和表观遗传学调控而发挥抗肿瘤作用，但硒的抗癌作用机制尚不十分明了。本项目将利用细胞和动物模型研究硒的作用，检测组蛋白去乙酰化酶蛋白水平及活性，并研究其降解机制；检测Nrf2通路的活化状态，研究抑制因子Keap1蛋白的降解及机制；证明硒可以加速组蛋白去乙酰化酶的降解过程，促进组蛋白的乙酰化，同时通过调控miR-200a-Keap1途径，引发Nrf2蛋白富集并入核，激活Nrf2通路，从而抑制肿瘤的发生。通过本项研究，我们试图阐释补充硒可以降低降低食管癌发病的分子机理，为食管癌的防治提供实验室依据。

我国是食管癌高发国，其死亡率居恶性肿瘤第四位，五年生存率较低。越来越多的证据表明硒作为一种人体必需的微量元素，可以降低食管癌的发病风险，但确切的分子机制仍未被完全阐明。在本研究中，我们发现甲基烯酸MSA能够下调Keap1的蛋白水平，上调Nrf2蛋白入核，从而活化Keap1/Nrf2 信号通路。究其机制发现MSA处理可上调食管癌细胞miR-200a的表达水平，而Keap1又是miR-200a的直接靶基因，且利用miR-200a抑制剂Antagomir-200a处理食管癌细胞可以削弱MSA诱导的KEAP1下调，进一步研究发现MSA诱导miR-200a高表达依赖于KLF4的高表达，KLF4可直接正向调控miR-200a。我们还利用食管癌细胞系（KYSE150、KYSE510）和动物模型研究MSA的抗肿瘤机制。发现MSA可以下调食管癌细胞系EGFR的蛋白水平却不影响其mRNA水平，利用miRNA芯片分析发现MSA可以上调miR-146a,有报道证实EGFR是miR-146a的直接靶基因且miR-146a抑制剂Antagomir-146a可以削弱MSA所诱导的EGFR下调。利用4NQO小鼠模型也证实MSA可以在体内明显抑制小鼠食管肿瘤的数目和肿瘤负荷，也可在体内下调EGFR表达。细胞水平实验表明MSA可以抑制EGFR下游信号通路进而影响IL-6的外泌。为了明确IL-6与MSA抑制食管癌发生间的关系，我们用4NQO诱导IL-6敲除小鼠进而观察小鼠食管肿瘤的发生情况。结果表明IL-6缺失不影响小鼠食管癌的发生，却能部分削弱MSA的抑瘤作用。免疫组化实验进一步证实MSA可通过IL-6进而影响肿瘤微环境中巨噬细胞和中性粒细胞的浸润。总之，我们研究证实MSA作为化学预防药物可通过KLF4/miR-200a/Keap1/Nrf2轴来调控食管癌细胞，还为MSA作为食管癌治疗的潜在药物提供了理论依据。