锌转运蛋白Slc39a5/Zip5在食管癌发生发展中的作用及机制研究

Esophageal cancer is a common cancer in China. The study showed that dietary zinc deficiency can dramatically promote the development of esophageal squamous cell carcinoma induced by the nitrosamine compounds. However, plasma zinc homeostasis covers up the individual zinc storage capacity, dietary zinc deficiency does not accurately reflect body zinc status. The study found that the zinc ion transport protein Slc39a5/Zip5 which is the major zinc ions regulation protein can be used as a monitor of body zinc status, but the relationship between Slc39a5/Zip5 and esophageal cancer has not yet been reported. I have been working at high risk area of esophageal cancer in China for nearly 20 years. A large number of esophageal cancer, precancerous lesions and normal epithelial tissues were collected during the time. Dr. Wang Fudi,our collaborator found Zip5 first in the world, is an international leader in the study field of zinc. We tested the Zip5 protein expression in esophageal cancer, precancerous lesions and normal tissues used immunohistochemistry.The result is Zip5 protein expression showed a gradually decreased from esophageal cancer tissue to precancerous lesions and normal epithelial tissue. Zip5 protein expression in esophageal cancer tissues was significantly higher than the cancer adjacent tissues. The study suggests that Zip5 play an important role in the esophageal cancer occurrence and development. The project intends to expore the function and molucular mechanisms of Slc39a5/Zip5 in the occurrence and development of esophageal cancer use SiRNDL TM ,ion mass spectrometry as well as gene knockout mouse model technology,to find the target of prevention and treatment of esophageal cancer.

食管癌是我国常见恶性肿瘤。研究表明膳食中锌缺乏促进了亚硝胺类化合物诱导的食管鳞癌的发生发展。但是血浆锌稳态掩盖了个体锌的储存量，膳食锌缺乏不能准确反映机体锌的状态。研究发现锌离子转运蛋白Slc39a5/Zip5是调控锌离子的主要蛋白，可作为机体锌状态的监视器，但其与食管癌关系尚未见报道。申请者长期工作在我国食管癌高发现场，收集了大量食管癌、癌前病变及正常上皮组织标本，合作单位Wang Fudi博士首先发现了Zip5，在锌的研究领域处于国际领先地位。前期我们利用免疫组化在食管癌各级病变组织检测Zip5蛋白的表达情况，发现其在食管癌、癌前病变、正常组织中的表达呈梯度降低，在食管癌组织中的表达显著高于癌旁组织，提示其在食管癌发生、发展中均具有重要作用。本项目拟利用小分子调控RNAi慢病毒系统、基因敲除小鼠模型等技术探讨Zip5在食管癌发生、发展中的作用及分子机制，寻找食管癌预防治疗新的靶点。

本研究首次针对锌转运蛋白Slc39a5/ ZIP5进行了体内外研究，发现ZIP5可能通过COX2、E-cadherin信号通路发挥促癌作用，ZIP5有望成为高发区精准诊疗的新靶点。.1.从细胞水平探讨了ZIP5在食管癌进展中的作用:免疫组化、WESTERN BLOT和qRT‑PCR结果显示ZIP5在人食管癌组织中高表达；构建了ZIP5稳定低表达食管癌细胞系KYSE170K及对照组细胞系KYSE170S；MTT、CCK-8、Transwell法和流式细胞术提示ZIP5表达下调能降低细胞的增殖、转移和侵袭能力；分别用WESTERN BLOT和qRT‑PCR检测与细胞增殖、转移、周期相关癌基因，提示ZIP5可能是通过影响COX2，CyclinD1，E-Cadherin的表达来发挥癌基因作用。.2.利用裸鼠移植瘤模型探讨ZIP5在食管癌中的作用: 成功建立了KYSE170K和KYSE170S裸鼠移植瘤模型；数据显示实验组移植瘤生长速度显著慢于对照组，瘤体积明显小于对照组；qRT‑PCR、WESTERN BLOT和免疫组化结果显示实验组中COX2表达下降、E-cadherin表达升高。.3.利用ZIP5基因敲除小鼠探讨ZIP5食管癌进展中的作用：成功构建ZIP5基因敲除小鼠；用4NQO饮水法建立了ZIP5基因敲除小鼠食管癌模型；用不同锌水平饲料喂养ZIP5基因敲除小鼠；用HE染色及免疫组化方法检测各组小鼠食管病理情况；分别用qRT‑PCR、WESTERN BLOT和免疫组化检测ZIP5及癌相关基因的表达水平。.4.锌相关miRNA在食管癌高发区作为食管癌预后标记物的研究：比较来自食管癌高发区、低发区的食管癌患者及健康志愿者的血清锌水平，结果显示食管癌患者的血清锌水平低于健康人群，食管癌高发区的健康人群血清锌水平与患者相近，显著低于低发区的健康人群；以不同锌水平培养食管癌细胞系细胞检测miRNA表达水平，结果显示：miR-21，miR-31，miR-93，miR-375的表达受锌水平影响；检测88例食管癌患者miRNA的表达水平，并对其进行随访，结果显示miR-21高表达和miR-375低表达的患者预后较差。.5.探讨了食管癌高发区预后相关的血清指标：结果显示NLR是高发区患者明确的预后指标。.6.通过大数据分析明确了河北省特别是高发区食管癌流行规律。