Lee Jong Heun, Horak Christine E, Khanna Chand, Meng Zhaojing, Yu Li Rong, Veenstra Timothy D, Steeg Patricia S
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Cancer Res. 2008 Feb 1;68(3):639-44. doi: 10.1158/0008-5472.CAN-07-2632.
The role of Gemin5 in alternative mRNA splicing, tumor cell motility, and proteomic instability was investigated. Isotope Capture Affinity Tag proteomic analysis was conducted on MDA-MB-435 tumor cells transfected with either a control vector (C-100) or the Nm23-H1 metastasis suppressor (H1-177). Ingenuity pathway analysis revealed that RNA posttranscriptional processing was the most prominent class of differentially expressed proteins. Within this category, overexpression of Acinus1, Poly(a) binding protein, HNRPA2B1, Bop1, and Gemin5 was confirmed in less metastatic H1-177 cells. Overexpression of the latter four proteins was also observed in the lower metastatic antisense Ezrin transfectant of a murine osteosarcoma model system, confirming the general relevance of the trends. Gemin5, a component of the spliceosomal complex, was chosen for further study. Analysis of global mRNA splicing by SpliceArray chips revealed that 16 genes were differentially spliced in C-100 compared with H1-177 cells; transient transfection of gemin5 into C-100 cells restored the splice pattern to that of H1-177 cells. Alternative splicing patterns for the engulfment and cell motility 1 and thrombospondin 4 genes were confirmed by semiquantitative reverse transcription-PCR. Gemin5 overexpression coordinately reduced C-100 cell motility by 50%, and siRNA-mediated reduction of Gemin5 expression increased the motility of H1-177 cells by 2-fold (P < 0.004). The data provide the first demonstration that alterations in the expression of a spliceosome protein can effect both specific splicing events and tumor cell motility. The data also show that changes in mRNA splicing patterns accompany metastatic progression, which may contribute to proteome instability.
研究了Gemin5在可变mRNA剪接、肿瘤细胞运动性和蛋白质组不稳定性中的作用。对用对照载体（C - 100）或Nm23 - H1转移抑制因子（H1 - 177）转染的MDA - MB - 435肿瘤细胞进行了同位素捕获亲和标签蛋白质组分析。 Ingenuity通路分析显示，RNA转录后加工是差异表达蛋白中最突出的类别。在这一类别中，在转移能力较低的H1 - 177细胞中证实了Acinus1、聚腺苷酸结合蛋白、HNRPA2B1、Bop1和Gemin5的过表达。在小鼠骨肉瘤模型系统的低转移反义埃兹蛋白转染细胞中也观察到后四种蛋白的过表达，证实了这些趋势的普遍相关性。选择剪接体复合物的一个组分Gemin5进行进一步研究。通过SpliceArray芯片分析全局mRNA剪接，结果显示与H1 - 177细胞相比，C - 100中有16个基因发生了差异剪接；将gemin5瞬时转染到C - 100细胞中可使剪接模式恢复到H1 - 177细胞的水平。通过半定量逆转录 - PCR证实了吞噬和细胞运动性1基因以及血小板反应蛋白4基因的可变剪接模式。Gemin5的过表达协同降低了C - 100细胞运动性50%，而siRNA介导的Gemin5表达降低使H1 - 177细胞的运动性增加了2倍（P < 0.004）。这些数据首次证明剪接体蛋白表达的改变可影响特定的剪接事件和肿瘤细胞运动性。数据还表明，mRNA剪接模式的变化伴随转移进展，这可能导致蛋白质组不稳定。
