Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Heping District, Tianjin, China.
PLoS One. 2012;7(5):e34832. doi: 10.1371/journal.pone.0034832. Epub 2012 May 2.
BAG-1 is an anti-apoptotic protein that interacts with a variety of cellular molecules to inhibit apoptosis. The mechanisms by which BAG-1 interacts with other proteins to inhibit apoptosis have been extensively explored. However, it is currently unknown how BAG-1 expression is regulated at the molecular level, especially in cancer cells. Here we reported to clone a novel down-regulated BAG-1 expression gene named FLJ20420 using hBAG-1 promoter as a probe to screen Human Hela 5' cDNA library by Southernwestern blot. The FLJ20420 gene encodes a ∼26-kDa protein that is localized in both the cytoplasm and nucleus. We proved that FLJ20420 protein can specially bind hBAG-1 promoter region by EMSA in vivo and ChIP assay in vivo. Northern blot analysis revealed a low level of FLJ20420 transcriptional expression in normal human tissues (i.e., brain, placenta, lung, liver, kidney, pancreas and cervix), except for heart and skeletal muscles, which showed higher levels. Furthermore, enhanced FLJ20420 expression was observed in tumor cell lines (i.e., MDA468, BT-20, MCF-7, C33A, HeLa and Caski). Knockdown of endogenous FLJ20420 expression significantly increased BAG-1 expression in A549 and L9981 cells, and also significantly enhanced their sensitivity to cisplatin-induced apoptosis. A microarray assay of the FLJ20420 siRNA -transfectants showed altered expression of 505 known genes, including 272 upregulated and 233 downregulated genes. Finally, our gene array studies in lung cancer tissue samples revealed a significant increase in FLJ20420 expression in primary lung cancer relative to the paired normal lung tissue controls (p = 0.0006). The increased expression of FLJ20420 corresponded to a significant decrease in BAG-1 protein expression in the primary lung cancers, relative to the paired normal lung tissue controls (p = 0.0001). Taken together, our experiments suggest that FLJ20420 functions as a down-regulator of BAG-1 expression. Its abnormal expression may be involved in the oncogenesis of human malignancies such as lung cancer.
BAG-1 是一种抗凋亡蛋白，可与多种细胞分子相互作用抑制细胞凋亡。BAG-1 与其他蛋白相互作用抑制凋亡的机制已被广泛研究。然而，目前尚不清楚 BAG-1 的表达是如何在分子水平上受到调控的，特别是在癌细胞中。在这里，我们报道了使用 hBAG-1 启动子作为探针，通过 Southernwestern blot 从人 Hela 5'cDNA 文库中筛选到一个新的下调 BAG-1 表达的基因，命名为 FLJ20420。FLJ20420 基因编码一个约 26kDa 的蛋白，定位于细胞质和细胞核中。我们通过体内 EMSA 和 ChIP 实验证明，FLJ20420 蛋白可以特异性结合 hBAG-1 启动子区域。Northern blot 分析显示，FLJ20420 在正常人体组织（如脑、胎盘、肺、肝、肾、胰腺和宫颈）中的转录表达水平较低，除了心脏和骨骼肌组织表达水平较高。此外，在肿瘤细胞系（如 MDA468、BT-20、MCF-7、C33A、HeLa 和 Caski）中观察到 FLJ20420 的表达增强。内源性 FLJ20420 表达的敲低显著增加了 A549 和 L9981 细胞中 BAG-1 的表达，并显著增强了它们对顺铂诱导凋亡的敏感性。FLJ20420 siRNA 转染细胞的微阵列分析显示，505 个已知基因的表达发生改变，其中 272 个基因上调，233 个基因下调。最后，我们在肺癌组织样本中的基因阵列研究表明，与配对的正常肺组织对照相比，原发性肺癌中 FLJ20420 的表达显著增加（p=0.0006）。原发性肺癌中 FLJ20420 的表达增加与配对的正常肺组织对照相比，BAG-1 蛋白表达显著降低（p=0.0001）。综上所述，我们的实验表明，FLJ20420 作为 BAG-1 表达的下调因子发挥作用。其异常表达可能参与肺癌等人类恶性肿瘤的发生。
