组蛋白H3K4三甲基化在砷诱导人支气管上皮细胞恶性转化中的作用

Arsenic does not act as a classical carcinogen, and the mechanism by which arsenic induces cancers remains obscure. Therefore, epigenetic approach has become increasingly important in elucidating the mechanism of arsenic's carcinogenicity. In the previous study, we found that acute arsenite exposure increased the transcription activation mark H3K4 tri-methylation in human bronchial epithelial BEAS-2B cells. Since cell transformation assay has been widely used to elucidate the modes of action underlying carcinogenicity, we are going to transform BEAS-2B cells in this proposal by measuring anchorage-independent growth and tumorigenicity in nude mice following arsenite exposure. We will then measure H3K4 tri-methylation, the activities and expression levels of H3K4 methyltransferases and demethylases by Western blot, Real-Time PCR, and in vitro H3K4 methylation and demethylation assay in transformed BEAS-2B cells. In addition, we will knock down (shRNA) and overexpress (gene transfection) KDM5A and SMYD3 in BEAS-2B cells to investigate if they are involved in arsenite-increased H3K4 tri-methylation. Finally, we will be able to evaluate the role of H3K4 tri-methylation in arsenite-induced transformation of the cells by knock down and overexpression of KDM5A and SMYD3. This study will further elucidate the molecular mechanism by which arsenic induces cancers, and help to reduce the health risks associated with arsenic exposure.

砷不是典型的遗传毒性致癌物，其致癌机制仍不十分清楚，因而从表观遗传途径研究其致癌机制有重要意义。前期研究发现，在人支气管上皮细胞（BEAS-2B）中急性砷暴露引起转录活化的信号H3K4三甲基化增加。鉴于细胞恶性转化在探讨致癌机制中常被作为实验观察终点，本项目拟通过软琼脂克隆形成实验和裸鼠成瘤实验建立三价无机砷诱导BEAS-2B细胞恶性转化的模型，采用基因转染、shRNA、Western blot、实时荧光定量PCR等技术，研究恶性转化的细胞中H3K4三甲基化水平，及H3K4甲基转移酶和去甲基化酶的活性和表达水平；明确细胞中的H3K4甲基转移酶SMYD3和去甲基化酶KDM5A正常表达、增加或降低与砷改变H3K4三甲基化水平的关系；进而通过二者的高表达和低表达调节H3K4三甲基化，弄清H3K4三甲基化在砷诱导细胞恶性转化中的作用，为进一步阐明砷诱导肺癌的机制和控制砷暴露的健康风险提供科学依据。

砷不是典型的遗传毒性致癌物，其致癌机制仍不十分清楚，因而从表观遗传途径研究其致癌机制具有重要意义。本项目主要研究砷诱导人支气管上皮BEAS-2B细胞转化过程中对组蛋白H3K4三甲基化、H3K9二甲基化和H3K27三甲基化的作用及其机制，以及组蛋白甲基化的异常与细胞转化的关系。研究结果表明：砷染毒16周时诱导BEAS-2B细胞转化；砷诱导细胞转化过程中H3K4三甲基化、H3K9二甲基化和H3K27三甲基化水平显著降低；砷可通过降低H3K4甲基转移酶活性和增加去甲基化酶活性使H3K4三甲基化水平降低，或通过增加H3K9去甲基化酶活性使H3K9二甲基化水平降低，但砷在诱导细胞转化过程中对组蛋白去甲基化酶KDM5A和KDM3A蛋白的水平无影响。该结果表明H3K4三甲基化、H3K9二甲基化和H3K27三甲基化水平的改变可能在砷诱导细胞转化过程中起作用，该研究结果能够为进一步阐明砷诱导肺癌的机制和控制砷暴露的健康风险提供科学依据。