Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin, China.
Deparment of Cardiothoracic Surgery, Tianjin Medical University General Hospital, Tianjin, China.
Thorac Cancer. 2017 May;8(3):131-137. doi: 10.1111/1759-7714.12408. Epub 2017 Mar 13.
Epithelial to mesenchymal transition (EMT) is a complex and dynamic molecular event in lung cancer metastasis that has not yet been thoroughly investigated. EMT transcriptional factors, such as Snail, play a central role in regulation of the EMT process. In this study, we sought to identify an association between p300 and Snail in lung cancer, as well as the engagement of p300 in Snail acetylation.
We transfected p300 small interfering RNA into lung cancer cells to detect Snail and E-cadherin expression levels by real time-PCR. Immunoprecipitation assay was conducted to determine Snail acetylation in vivo. Bacteria-expressed Snail was purified to analyze Snail acetylation in vitro. We further mutated lysine 187 for identifying acetylated residue in Snail.
Snail transcription in lung cancer cells was repressed by p300 knockdown. E-cadherin expression was increased by transfection of p300 small interfering RNA in a dose-dependent manner. Immunoprecipitation and Western blot assay with anti-acetylated lysine antibody were used to confirm that Snail was acetylated by p300. A sequence coding snail gene was cloned into glutathione S-transferase-tagged vector and the fusion protein was purified using glutathione. We observed Snail acetylation in vitro by incubation of recombinant Snail and p300 histone acetyltransferase domain with acetyl coenzyme A. The reduced Snail acetylation level was related to lysine mutation at position 187 of Snail.
There was a correlation between Snail and p300 expressions in lung cancer. Moreover, p300 acetylates Snail both in vivo and in vitro, and K187 may be involved in this modification.
上皮间质转化（EMT）是肺癌转移中一个复杂而动态的分子事件，尚未得到彻底研究。EMT 转录因子如 Snail 在 EMT 过程的调控中起着核心作用。在这项研究中，我们试图确定 p300 与肺癌中的 Snail 之间的关联，以及 p300 参与 Snail 乙酰化。
我们将 p300 小干扰 RNA 转染到肺癌细胞中，通过实时 PCR 检测 Snail 和 E-钙黏蛋白的表达水平。免疫沉淀试验用于体内检测 Snail 乙酰化。细菌表达的 Snail 被纯化以分析体外的 Snail 乙酰化。我们进一步突变赖氨酸 187 以确定 Snail 中的乙酰化残基。
p300 敲低抑制了肺癌细胞中的 Snail 转录。p300 小干扰 RNA 的转染剂量依赖性地增加了 E-钙黏蛋白的表达。用抗乙酰化赖氨酸抗体进行免疫沉淀和 Western blot 分析证实了 Snail 被 p300 乙酰化。克隆编码 snail 基因的序列到谷胱甘肽 S-转移酶标记载体中，并使用谷胱甘肽纯化融合蛋白。我们通过将重组 Snail 和 p300 组蛋白乙酰转移酶结构域与乙酰辅酶 A 孵育，观察到体外的 Snail 乙酰化。Snail 乙酰化水平的降低与 Snail 第 187 位赖氨酸的突变有关。
肺癌中存在 Snail 和 p300 表达之间的相关性。此外，p300 在体内和体外都乙酰化了 Snail，并且 K187 可能参与了这种修饰。
