circ-0088510/miR-181a调控Bclaf1表达在亚硝胺诱导人食管上皮细胞恶性转化中的作用及机制研究

Esophageal cancer is an environmentally-related tumor. It’s supposed that exposure of nitrosamines is an important risk factor, but he underlying mechanism of nitrosamine-induced esophageal cancer remains poorly understood. We found that increased expression of Bclaf1 occurred in the early stage of nitrosamine-induced human esophageal cell carcinogenesis, and involved in cell proliferation after carcinogenesis. Also, we found that inhibitor miR‐181a increased Bclaf1 mRNA upon exposure to nitrosamine. Bioinformatics analysis identified circ-0088510 acts as a sponge of miR-181a, which highly suggested that circ-0088510/miR-181a may regulate the expression of Bclaf1 involve in nitrosamine-induced malignant transformation of esophageal epithelial cells. In this project, nitrosamine-induced malignant transformation cell line models and esophageal cancer animal models for parallel comparison combined stable transfection method and RNA sequencing analysis to explore the function and specific mechanism of Bclaf1.The circ-0088510 and miR-181a loss and gain of function will be achieved by siRNA and a plasmid system to analyze how circ-0088510/miR-181a modulates the expression of Bclaf1. The CRISPR-Pro will be employed to establish a conditional knockout animal model of Bclaf1 gene. We will focus on the role of circ-0088510/miR-181a regulates the expression of Bclaf1 plays in cell proliferation to elucidate the molecular mechanism of nitrosamine-induced esophageal cancer and provide new insight on early prevention of esophageal cancer.

食管癌是环境相关性肿瘤，亚硝胺作为重要危险因素，其诱发食管癌的发病机制仍不明确。前期发现，亚硝胺诱导癌变早期中，Bclaf1水平升高，且参与癌变后细胞的增殖；亚硝胺作用下，抑制miR-181a可上调Bclaf1 mRNA水平；生信分析显示circ-0088510与miR-181a靶向结合，高度提示circ-0088510/miR-181a可能调控Bclaf1参与亚硝胺诱导的恶性转化。基于此，拟建立亚硝胺诱导细胞恶性转化和动物食管癌模型，采用稳定干扰技术探讨Bclaf1功能和RNA测序研究Bclaf1作用机制；以siRNA和质粒转染技术分析circ-0088510/miR-181a对Bclaf1调节作用；以CRISPR-Pro建立条件性敲除Bclaf1小鼠模型，从体内外阐明circ-0088510/miR-181a调控Bclaf1表达在恶性转化中的作用及机制，为食管癌的早期预防提供新依据。

食管癌是环境相关性肿瘤，亚硝胺类物质暴露是重要危险因素之一。但是，迄今为止其发生机制尚未完全明了，无疑不能充分地认识亚硝胺所致食管癌的生物学基础，也未能对食管癌的防治产生重要影响。因此，亚硝胺诱导的癌前病变及分子机制值得关注。亚硝胺诱导的食管癌的机制由多水平、多环节相互调控的网络组成，探讨亚硝胺致癌的可能分子机制对设计新的干预策略不无裨益。本研究以亚硝胺诱导人食管上皮细胞恶性转化细胞模型，探讨BCL-2相关转录因子1（Bcl-2- associated transcription factor 1，Bclaf1）在亚硝胺诱导食管癌过程中的上下游机制。建立了gain-of-function及loss-of-function功能研究体系，分析了Bclaf1表达改变后，细胞增殖和DNA复制活性等细胞生物学性状改变，以及3D细胞培养成团能力与裸鼠体内成瘤实验等细胞恶性表型的变化，结果表明Bclaf1在亚硝胺诱导人食管上皮细胞恶性转化过程中具有重要作用。以细胞免疫荧光和染色质提取等实验手段，检测出Bclaf1主要定位于恶性转化细胞的染色质。通过ATAC测序技术和生物信息学等方法，发现Bclaf1调控染色质的开放性介导转录重编程，并筛选出Bclaf1致癌作用的重要下游分子POLR2A。以ChIP和EMSA等实验方法，分析了Bclaf1调节POLR2A的表达促进染色质开放性的机制。以RNA干扰和过表达等技术，分析miR-181a对Bclaf1转录后的调节作用。收集食管癌临床样本和利用组织芯片，检测出Bclaf1在食管癌组织中高表达，结合公共数据库的分析，发现Bclaf1高表达与预后不良密切相关，提示Bclaf1具有作为食管癌的早期预警分子的潜在性。本项目解析Bclaf1在恶性转化过程中的生物学功能以及Bclaf1调控转录重编程参与亚硝胺诱导人食管上皮细胞恶性转化的分子机制，不仅有助于扩展对亚硝胺致癌效应的认识，也为食管癌的防治研究提供有益的补充。