Department of Gastroenteropancreatic Surgery, the Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510120, China.
Chin Med J (Engl). 2011 Nov;124(22):3778-85.
Considerable evidence suggests that phosphatase of regenerating liver-3 (PRL-3) plays multiple roles in cancer metastasis; however, the molecular mechanisms remain largely unknown. The aim of this study was to identify proteins associated with PRL-3-promoted colon cancer metastasis, by comparative proteomic analysis.
Proteomes of human colon cancer LoVo cells transfected with PRL-3 gene (LoVo-PRL-3) or empty vector PAcGFP-C3 (LoVo-control) were compared using 2D gel electrophoresis. Proteins that varied significantly in concentration were selected and identified using mass spectrometry. Expression of translationally controlled tumor protein (TCTP) mRNA and protein in LoVo-PRL-3 and LoVo-control cells was detected by real-time PCR and Western blotting. Small interfering RNA (siRNA) targeting TCTP was used for silencing TCTP expression in LoVo-PRL-3 cells. Functional significance of TCTP in PRL-3-promoted colon cancer cell proliferation, migration and invasion was investigated by Cell Counting Kit-8 assay and transwell chamber.
Seventeen proteins displaying significant and reproducible differences between LoVo-PRL-3 and LoVo-control cells were identified. Ten proteins were upregulated and seven were downregulated in LoVo-PRL-3 cells when compared with LoVo-control cells. Eight identified proteins are associated with distinct steps of tumor metastasis: ubiquitin-like protein ISG15, interleukin-18, TCTP, serpin B5, annexin A3, macrophage-capping protein, ATP-dependent RNA helicase DDX3X, and cathepsin D. Real-time PCR and Western blotting results showed that both TCTP mRNA and protein were significantly increased in LoVo-PRL-3 cells compared to LoVo-control cells. Transfection with TCTP siRNA significantly reduced the expression of both mRNA and protein levels of TCTP in LoVo-PRL-3 cells. Knockdown of TCTP by siRNA inhibited PRL-3-promoted proliferation, migration and invasion of LoVo-PRL-3 cells.
Our results imply that TCTP might be a mediator of PRL-3-promoted proliferation, migration and invasion of human colon cancer cells.
大量证据表明，肝再生磷酸酶-3（PRL-3）在癌症转移中发挥多种作用；然而，其分子机制在很大程度上仍然未知。本研究旨在通过比较蛋白质组学分析，鉴定与 PRL-3 促进结肠癌转移相关的蛋白质。
使用二维凝胶电泳比较转染 PRL-3 基因（LoVo-PRL-3）或空载体 PAcGFP-C3（LoVo-control）的人结肠癌 LoVo 细胞的蛋白质组。选择浓度差异显著的蛋白质，并使用质谱法进行鉴定。通过实时 PCR 和 Western blot 检测 LoVo-PRL-3 和 LoVo-control 细胞中转译控制肿瘤蛋白（TCTP）mRNA 和蛋白的表达。用靶向 TCTP 的小干扰 RNA（siRNA）沉默 LoVo-PRL-3 细胞中的 TCTP 表达。通过细胞计数试剂盒-8 测定和 Transwell 室实验研究 TCTP 在 PRL-3 促进结肠癌细胞增殖、迁移和侵袭中的功能意义。
在 LoVo-PRL-3 和 LoVo-control 细胞之间鉴定出 17 种显示出显著且可重复差异的蛋白质。与 LoVo-control 细胞相比，LoVo-PRL-3 细胞中有 10 种蛋白质上调，7 种蛋白质下调。鉴定出的 8 种蛋白质与肿瘤转移的不同步骤有关：泛素样蛋白 ISG15、白细胞介素-18、TCTP、丝氨酸蛋白酶抑制剂 B5、膜联蛋白 A3、巨噬细胞封顶蛋白、ATP 依赖的 RNA 解旋酶 DDX3X 和组织蛋白酶 D。实时 PCR 和 Western blot 结果显示，与 LoVo-control 细胞相比，LoVo-PRL-3 细胞中的 TCTP mRNA 和蛋白表达均显著增加。用 TCTP siRNA 转染显著降低了 LoVo-PRL-3 细胞中 TCTP 的 mRNA 和蛋白水平的表达。用 siRNA 敲低 TCTP 抑制了 PRL-3 促进的 LoVo-PRL-3 细胞的增殖、迁移和侵袭。
我们的结果表明，TCTP 可能是 PRL-3 促进人结肠癌细胞增殖、迁移和侵袭的介质。
