Scinderin基因调控前列腺癌细胞增殖的相关分子机制研究

There is currently no cure for androgen independent prostate cancer (AIPC). It is urgent to explore its pathogenesis and develop new molecularly targeted drugs of potential clinical value. In our previous study, significantly higher expression levels of Scinderin protein were found in prostate cancer samples than in normal tissues adjacent to prostate cancer tissues, and the expression levels of Scinderin protein was significantly related to the relapse and metastasis of prostate cancers. Moreover, Scinderin gene was involved in the regulation of proliferation of prostate cancer cells. But the exact mechanism of its action is unclear. Therefore, based upon our previous results, the present study was designed. In the present investigation, lentivirus-mediated RNA interference technology was used to achieve long term silence of Scinderin gene in prostate cancer cells (PC-3). Affymetrix GeneChips, Proteomic technologie and bioinformatic analysis were also used to explore the molecular mechanism by which Scinderin regulates the proliferation of prostate cancer cells, and to find the related signaling pathway and downstream molecule of Scinderin gene. Furthermore, up-regulation or down-regulation of some candidate molecules were performed, and their effects on the proliferation of prostate cancer cell proliferation were observed. Based on these results, we will elucidate the roles of Scinderin gene and its downstream candidate molecules in the proliferation of androgen independent prostate cancer cells in molecular level, which will provide theoretical proofs for elucidating mechanism of cell proliferation of androgen independent prostate cancer and developing new molecular targets.

雄激素非依赖性前列腺癌是一种不可治愈的疾病，迫切需要研究其发病机制，开发具有临床应用价值的分子靶向药物。我们前期研究中发现Scinderin基因在前列腺癌组织中高表达，与前列腺癌的复发及转移密切相关，参与调控肿瘤细胞恶性增殖，但具体作用机制不明。因此，我们以前列腺癌细胞系PC-3为研究对象，首先，应用RNAi联合慢病毒技术下调PC-3细胞中Scinderin基因的表达，并利用基因芯片技术、蛋白质组学技术及生物信息学技术初步探讨沉默Scinderin基因抑制PC-3细胞增殖的分子机制，随后，筛选、验证出相关信号通路和下游分子；最后，上调或下调上述与PC-3细胞增殖相关的下游分子的表达水平，观察其对PC-3细胞增殖的影响，从分子水平阐明这些分子在介导雄激素非依赖性前列腺癌细胞恶性增殖中的可能机制。为阐明雄激素非依赖性前列腺癌细胞增殖的分子机制、调控网络和寻找新的治疗靶点提供理论依据和实践基础。

本项目研究发现SCIN在前列腺癌组织中显著高表达且与前列腺癌患者的分化程度有关（p<0.05）。在细胞模型和动物模型都验证了，沉默SCIN导致前列腺癌细胞增殖抑制。通过表达谱芯片分析鉴定了前列腺癌细胞株中SCIN沉默介导的下游mRNA表达谱。总计筛选到5067个基因在SCIN基因沉默组和对照组之间有显著差异（p<0.05）。在众多SCIN下游基因中，我们关注到了表皮生长因子（EGFR）分子。在动物模型中也验证了SCIN基因沉导致EGFR蛋白下调。向SCIN基因沉默的细胞株中加入FGF因子，使得EGFR的磷酸化和内化作用被诱导，发现本来SCIN基因沉默导致的前列腺癌细胞增殖被抑制的效应，在加入EGF之后被削弱了。与此同时，SCIN沉默本来导致磷酸化MEK蛋白，磷酸化AKT蛋白、磷酸化ERK蛋白的表达水平显著降低，但加入EGF刺激之后，3种磷酸化蛋白的表达水平被回复。此外，SCIN基因沉默能够导致细胞凋亡有关的蛋白显著上调，包括裂解的PARP、 caspase-3和caspase-9. .通过表达谱芯片筛选，我们同时关注到PYCR1和MYO6作为SCIN的下游调节基因，与SCIN的表达具有正相关性。在前列腺癌组织中，均检测到了PYCR1蛋白和MYO6蛋白的高表达，且它们的表达均与患者的Gleason 评分显著相关 (P<0.05)。沉默MYO6基因或者PYCR1基因表达均能够显著抑制前列腺癌细胞增殖，并诱导凋亡。沉默MYO6基因表达可导致磷酸化ERK和磷酸化AKT蛋白的表达下调。沉默PYCR1基因表达可导致裂解的PARP蛋白和caspase-3 蛋白显著上调。综上，研究发现SCIN和PYCR1的表达与SCIN具有正相关性，也在前列腺癌中扮演着促进肿瘤增殖的作用。.总体上，本项目揭示了SCIN在前列腺癌恶性进展中的重要作用。SCIN的下游调节网络纷繁复杂，我们从EGFR这一重要的受体信号转导的蛋白入手，发现了EGFR-ERK和EGFR-AKT 通路均参与调节了SCIN对前列腺癌细胞增殖和凋亡的影响。我们的研将为前列腺癌的基因治疗提供潜在的分子靶标。