Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan Town, Miaoli, Taiwan, Republic of China.
PLoS One. 2013;8(3):e58227. doi: 10.1371/journal.pone.0058227. Epub 2013 Mar 11.
The expression of polypyrimidine tract-binding protein (PTB) is up-regulated in many types of cancer. Here, we studied the role of PTB in the growth of non small cell lung cancer cells. Data showed that PTB overexpression inhibited the growth of H1299 cells at least by inhibiting DNA synthesis. Quantitative real-time PCR and Western blot analyses showed that PTB overexpression in H1299 cells specifically induced the expression of p19(Ink4d), an inhibitor of cyclin-dependent kinase 4. Repression of p19(Ink4d) expression partially rescued PTB-caused proliferation inhibition. PTB overexpression also inhibited the growth and induced the expression of p19(Ink4d) mRNA in A549 cells. However, Western blot analyses failed to detect the presence of p19(Ink4d) protein in A549 cells. To address how PTB induced p19(Ink4d) in H1299 cells, we showed that PTB might up-regulate the activity of p19(Ink4d) gene (CDKN2D) promoter. Besides, PTB lacking the RNA recognition motif 3 (RRM3) was less effective in growth inhibition and p19(Ink4d) induction, suggesting that RNA-binding activity of PTB plays an important role in p19(Ink4d) induction. However, immunoprecipitation of ribonuclearprotein complexes plus quantitative real-time PCR analyses showed that PTB might not bind p19(Ink4d) mRNA, suggesting that PTB overexpression might trigger the other RNA-binding protein(s) to bind p19(Ink4d) mRNA. Subsequently, RNA electrophoretic mobility-shift assays revealed a 300-base segment (designated as B2) within the 3'UTR of p19(Ink4d) mRNA, with which the cytoplasmic lysates of PTB-overexpressing cells formed more prominent complexes than did control cell lysates. Insertion of B2 into a reporter construct increased the expression of the chimeric luciferase transcripts in transfected PTB-overexpressing cells but not in control cells; conversely, overexpression of B2-containing reporter construct in PTB-overexpressing cells abolished the induction of p19(Ink4d) mRNA. In sum, we have shown that PTB plays as a negative regulator in H1299 cell proliferation at least by inducing p19(Ink4d) expression at transcriptional and post-transcriptional levels.
多嘧啶结合蛋白（PTB）的表达在许多类型的癌症中上调。在这里，我们研究了 PTB 在非小细胞肺癌细胞生长中的作用。数据表明，PTB 的过表达至少通过抑制 DNA 合成来抑制 H1299 细胞的生长。定量实时 PCR 和 Western blot 分析表明，PTB 在 H1299 细胞中的过表达特异性诱导了细胞周期蛋白依赖性激酶 4 抑制剂 p19(Ink4d)的表达。p19(Ink4d)表达的抑制部分挽救了 PTB 引起的增殖抑制。PTB 的过表达也抑制了 A549 细胞的生长，并诱导了 p19(Ink4d)mRNA 的表达。然而，Western blot 分析未能在 A549 细胞中检测到 p19(Ink4d)蛋白的存在。为了解决 PTB 如何在 H1299 细胞中诱导 p19(Ink4d)的问题，我们表明 PTB 可能上调 p19(Ink4d)基因（CDKN2D）启动子的活性。此外，缺乏 RNA 识别基序 3（RRM3）的 PTB 在生长抑制和 p19(Ink4d)诱导方面的效果较差，表明 PTB 的 RNA 结合活性在 p19(Ink4d)诱导中发挥重要作用。然而，核糖核蛋白复合物的免疫沉淀加上定量实时 PCR 分析表明，PTB 可能不结合 p19(Ink4d)mRNA，这表明 PTB 的过表达可能触发其他 RNA 结合蛋白与 p19(Ink4d)mRNA 结合。随后，RNA 电泳迁移率变动分析显示，p19(Ink4d)mRNA 的 3'UTR 内存在一个 300 碱基的片段（命名为 B2），与对照细胞裂解物相比，过表达细胞的细胞质裂解物形成了更明显的复合物。将 B2 插入报告构建体中，增加了转染过表达细胞中嵌合荧光素酶转录物的表达，但在对照细胞中没有；相反，在过表达细胞中过表达含有 B2 的报告构建体，可消除对 p19(Ink4d)mRNA 的诱导。总之，我们已经表明，PTB 至少通过在转录和转录后水平诱导 p19(Ink4d)的表达，在 H1299 细胞增殖中发挥负调节作用。
