脂筏介导ZIP4调控STAT3参与胰腺癌发生发展的实验研究

Pancreatic ductal adenocarcinoma (PDA) is the most malignant cancer resulting in more than 250,000 deaths per year worldwide. Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids. The structure of lipid rafts is dynamic, resulting in an ever-changing content of both lipids and proteins. These highly dynamic raft domains are essential in signal transduction and also form sorting platforms for targeted protein traffic. Non-constitutive protein components of rafts fluctuate dramatically in cancer with impacts on cell proliferation, signaling, protein trafficking, adhesion and apoptosis. Several anti-cancer drugs are able to suppress growth and induce apoptosis of tumor cells through alteration of lipid raft contents via disrupting lipid raft integrity.The zinc transporter ZIP4 is overexpressed in most pancreatic cancer and is a malignant factor in cancer progression. And recent studies have shown that pancreatic cancer cells expressing high levels of ZIP4 exhibit greater in vitro cell proliferation and in vivo tumor progression. Silencing of ZIP4 inhibits pancreatic cancer growth and significantly increases the survival rate of nude mice with pancreatic cancer xenografts . However, the mechanisms through which ZIP4 promotes cancer progression are largely unknown. ZIP4 is a membrane protein, and our preliminary data showed that ZIP4 is associated with lipid rafts.Therefore, it is of great interest to investigate how lipid rafts mediate ZIP4 pathway in pancreatic cancer, and how to target lipid rafts and ZIP4 pathways to treat pancreatic cancer.

胰腺癌是常见且恶性程度极其高的肿瘤，每年世界范围内有25万患者死于该病。脂筏（lipid rafts）是细胞膜上的功能微区，富含胆固醇和鞘脂。脂筏的结构是动态变化的，使其脂质和蛋白含量时刻在变化。脂筏的动态变化对信号转导起重要作用，并且起到信号平台的作用，有益于靶蛋白的转运。脂筏结构中非结构蛋白的动态变化对肿瘤细胞的增殖、信号传导、蛋白转运、粘附和凋亡起到十分重要作用。有研究发现几种抗肿瘤药物通过改变脂筏内成分破坏其整体性，从而抑制肿瘤细胞生长和诱导其凋亡。ZIP4在大多数胰腺癌组织中高表达，体内体外实验均证实其参与胰腺癌的发生发展。近期研究发现沉默ZIP4表达可抑制胰腺癌的的生长，明显增加胰腺癌裸鼠的生存率。但是其具体机制还未阐明。ZIP4表达的蛋白为膜蛋白，初步实验证实其存在于脂筏结构中，是否脂筏介导ZIP4信号传导参与胰腺癌的发生发展，其具体机制如何，答案均需进一步深入研究。

胰腺癌是恶性程度极其高，预后很差。目前，胰腺癌发病机制仍未完全阐明。脂筏是细胞膜上富含胆固醇和鞘脂的功能微区，其结构内脂质和蛋白含量呈动态变化。锌转运蛋白ZIP4 在胰腺癌组织中呈高表达，体内体外实验均证实其在胰腺癌的发生发展中发挥重要作用。脂筏参与肿瘤细胞增殖、信号传导、蛋白转运、粘附和凋亡等过程。ZIP4的功能是否与定位于脂筏结构内有关尚无相关报道。为此，本课题旨在明确胰腺癌细胞中 ZIP4与脂筏的关系和脂筏介导ZIP4信号通路对胰腺癌生物学特性的影响并探索其具体机制。本研究通过蔗糖密度梯度离心法成功提取胰腺癌稳定细胞株MIA-ZIP4的脂筏，Western Blot实验结果显示 ZIP4蛋白位于脂筏结构中。分别用非离子型去垢剂methyl-β-cytodextrin 和octyl-β-glucoside处理胰腺癌细胞株 MIA-ZIP4后，再通过蔗糖密度梯度离心法提取胰腺癌稳定细胞株 MIA-ZIP4的脂筏，Western Blot实验结果显示非离子型去垢剂可打破ZIP4 蛋白相关脂筏。CSS-palm 3.0软件预测ZIP4共有15个棕榈化位点，结合ZIP4预测的跨膜结构，我们初步推测，有6个（Cys 338、Cys 340、Cys 350、Cys 353、Cys 436、Cys 535、Cys 603）位于ZIP4跨膜区域或近胞浆侧的Cys为最可能的棕榈化修饰位点，也就是ZIP4定位于脂筏结构的重要机制。任何一个上述位点点突变（Cys→Ser）后都会影响ZIP4蛋白定位于脂筏。建立稳定转染ZIP4突变细胞株，突变株细胞增殖和侵袭能力显著低于MIA-ZIP4细胞。肿瘤细胞注射构建裸鼠胰腺癌原位移植瘤，突变细胞株组不同时间点裸鼠的成瘤率、肿瘤大小、肝肺转移发生率均显著低于对照组。本课题研究成果将为后续深入研究胰腺癌发病机制奠定基础，为靶向治疗提供新的靶点。