Amplification and overexpression of vinculin are associated with increased tumor cell proliferation and progression in advanced prostate cancer

We analyzed the 10q22 amplification in advanced prostate cancer and subjected the genes located in the common amplified region to an RNAi screen. We found vinculin as the most promising candidate gene and analyzed its protein expression on more than 400 prostate cancers by using the tissue micrarray (TMA) technology. We discovered a strong correlation between amplification of the vinculin gene and increased protein expression. Further, vinculin protein expression was strongly associated with increased tumor cell proliferation. We isolated DNA from four 10q22 amplified cell lines (PC-3, MFM-223, SK-BR-3, ME-180). Genomic DNA was digested with the restriction enzymes RSA I / ALU I and then labeled with Cy5 using a Klenow-based commercial kit (Invitrogen). Each sample was hybridized with a pooled normal (46,XX) reference (Promega) to Agilent 244k CGH arrays. This experiment allowed us to define the common amplified region of the 10q22 amplification.

本研究针对晚期前列腺癌中的10q22扩增（10q22 amplification）展开分析，并对定位于共同扩增区域内的基因开展了RNAi筛选（RNAi screen）。我们筛选得到纽蛋白（vinculin）作为最具潜力的候选基因，随后采用组织微阵列（tissue microarray，TMA）技术，对400余例前列腺癌样本进行了纽蛋白的蛋白质表达分析。结果显示，纽蛋白基因的扩增与该蛋白的表达上调存在显著相关性。进一步研究表明，纽蛋白的蛋白质表达水平与肿瘤细胞增殖能力增强密切相关。我们从4株携带10q22扩增的细胞系（PC-3、MFM-223、SK-BR-3、ME-180）中提取了基因组DNA，使用限制性内切酶（restriction enzymes）RSA I与ALU I对基因组DNA进行酶切，随后采用基于Klenow酶的商用试剂盒（Klenow-based commercial kit，Invitrogen）以Cy5完成荧光标记。将每份待测样本与核型为46,XX的混合正常参考样本（Promega）共同杂交至安捷伦244k比较基因组杂交芯片（Agilent 244k CGH arrays）。通过本实验，我们明确了10q22扩增的共同扩增区域。