中文摘要
LAPTM4B是2000年克隆到的肿瘤相关新基因。研究发现,该基因启动子区域存在着*1和*2型二种多态性,该区含有转录因子AP-4,CREB1等转录因子结构域。LAPTM4B在乳腺癌和肝癌中明显高表达,这是否与启动子区的多态性和转录调节有关,为了探索其机理,本课题从该基因启动子多态区入手,以AP-4为主要靶点,通过对乳癌和肝癌细胞和癌组织检测LAPTM4B和AP-4表达水平,应用凝胶迁移阻滞实验检测LAPTM4B和AP-4结合能力及特异性,应用分子定点突变技术构建AP-4结合LAPTM4B多态区突变表达载体,转染经筛选的癌细胞,应用PCR、ELISA、Western blot等分子细胞生物学技术,检测野生型和突变型对荧光表达的影响。进一步通过细胞增殖、迁移侵袭、细胞周期和裸鼠致瘤实验检测对细胞生物学行为的作用,这会明确其转录调控作用,揭示其在乳癌和肝癌发展中的作用机制,提供新标志。
英文摘要
Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a novel cloned cancer-associated gene. There are two alleles of the gene, LAPTM4B*1 and LAPTM4B*2, which contain binding sites of transcription factor AP-4 and CREB1 etc. LAPTM4 is overexpressed in the breast cancer and liver cancer cells, also its transcript is up-regulated in various types of solid tumors. However, its transcriptional regulation mechanism is still unclear. To investigate the mechanism of transcriptional regulation of LAPTM4B in human cancer cells, we try to measure expression levels of LAPTM4B and AP-4 in breast cancer and liver cancer cells and tissues, binding ability of LAPTM4B polymorphism region with AP-4 by electrophoretic mobility shift assay and super-shift and RNA interference experiments, construct a series of luciferase reporter mutation constructs of LAPTM4B polymorphism region for AP-4 binding by site-directed mutation technique, which will be transfected to screened human cancer cells to determine the transcriptional activities of different promoter mutated regions, and check the report vector fluorescence expression by PCR, Northern blot、ELISA、Western blot molecule technology. Furthermore, the cancer cells will be tested by cell biology methods, such as MTT, flow cytometry, infiltrate, nude mouse to indentify cell proliferation, infiltrate ability, cell cycle and tumorigenesis. This will clarify that what kinds of roles AP-4 can play in LAPTM4B transcriptional regulation in human breast cancer and liver cancer cells, it will also indicate that significance between the interaction of LAPTM4B and AP-4 in the cancer occurrence and progression. It will be an important foundation for discover a novel tumor marker.
