SEI1-ILEI-EMT与卵巢癌发生及转移的关系研究

Mounting evidence suggests that a variety of oncogenes and cytokines are involved in the initiation and progression of epithelial-derived malignancies. Indeed, the interaction between oncogenes and cytokines can induce cell polarity change, cell malignant transformation, invasion and metastasis in cancer. In our previous study, we cloned and characterized an oncogene, SEI-1, in ovarian cancer. The oncogenic function of SEI-1 was associated with chromosome instability. When expressing SEI-1 in immortalized normal ovarian epithelial cell line HOSE6-3, we found it could induce oncogenic transformation of the HOSE6-3 cells. After array-CGH and microarray analysis, we found that the SEI-1 gene could notably induce DNA copy number gain and mRNA overexpression of a cytokine, ILEI. Our preliminary data also showed that ILE1 was significantly overexpressed in ovarian tumor tissues compared to normal ovarian tissues. Recent studies showed that the ILE1 gene is a newly discovered epithelial-mesenchymal transition (EMT) - inducing factor. Here, we hypothesize that SEI-1-ILEI-EMT is a novel oncogenic signaling pathway involved in the development and progression of ovarian cancer. The objectives in the present proposal will be achieved through the following interrelated specific aims: 1) to investigate the relationship between ILEI gene and genesis, metastasis of ovarian cancer; 2) to investigate the molecular mechanism of EMT induced by ILEI in these process; and 3) to investigate the mechanisms of the combined action between ILEI and SEI-1 responsible to EMT induction and promotion of tumorigenicity and metastasis of ovarian cancer. A series of experiments will be used in the present study, including lentiviral transfection, RNAi, gene function analysis, nude mice experiments in vivo, clinical association study with tissue microarray (TMA). This proposed study will help to understand the molecular basis for ovarian cancer development and provide new molecular targets for this deadly cancer.

前期研究发现，癌基因SEI-1可使人卵巢永生化上皮细胞HOSE6-3致瘤，且伴有ILEI基因（新发现的EMT诱导因子）扩增及过表达，说明SEI-1-ILEI-EMT可能是诱导卵巢癌发生及转移的新机制。本研究中，我们将通过ILEI慢病毒载体转染HOSE细胞，体外及裸鼠体内实验检测其致瘤性、转移性的变化及EMT的发生；同时转染卵巢癌细胞系，体外基因功能及裸鼠体内实验检测ILEI高表达后，细胞生物学特性及转移能力的改变，EMT发生及其分子机理。应用RNAi分别敲除ILEI、SEI-1的表达，基因功能分析、裸鼠体内实验探讨二者的协同作用。免疫荧光、免疫组化分析临床组织标本中ILEI的亚细胞定位及与SEI-1和肿瘤临床病理特征之间的关联。本研究将阐明SEI-1、ILEI通过协同作用，诱导EMT，促进卵巢癌发生及转移的分子机理，确认卵巢癌发生及转移相关的分子标记物，为肿瘤基础研究及临床治疗提供新靶标。

ILEI，即 FAM3C基因，研究发现，该基因通过诱导 EMT发生，在上皮源性肿瘤发生及转移过程中发挥重要作用，是一个新的 EMT诱导因子。本项目在前期工作的基础上，拟探讨 ILEI 与卵巢癌发生、发展及转移的关系，研究将有利于我们更深入了解卵巢癌发生发展及转移的分子机制并将提供一个理想的预防和治疗的新靶点。本项目中，我们首先对临床EOC肿瘤样本以及组织芯片中FAM3C的表达进行筛查，发现FAM3C在EOC组织中明显高表达（P<0.001），且与肿瘤的转移具有相关性（P<0.05）。应用体外构建FAM3C正、反义稳定转染的EOC细胞株，发现FAM3C可以促进EOC细胞迁移、侵袭，并可以诱导EOC细胞发生EMT。进一步在体内构建EOC裸鼠体内原位移植瘤模型，证实FAM3C可以促进EOC细胞体内的侵袭转移及EMT。通过特定生物学通路相关芯片对FAM3C促进EOC发生发展的分子机制进行筛查，并通过Real-Time PCR、Western Blot、免疫荧光等技术对芯片结果进行验证和进一步的分析，发现FAM3C是通过激活AKT/GSK3β/β-catenin通路轴，进而诱导EMT的发生。应用β-catenin的抑制剂可以降低EOC细胞的迁移和侵袭能力，下调JUN、MMP2、SPARC等通路分子，并且可使EMT发生逆转。此外，前期研究发现，癌基因SEI-1是ILEI（FAM3C）的上游靶基因，因此我们继续对SEI-1在卵巢癌中的功能进行研究。发现SEI1在临床卵巢癌样本中高表达，且其表达程度与临床肿瘤分期呈正相关。过表达SEI1基因可诱导基因组不稳定，增加DNA链断裂。同时发现SEI1在细胞核内与γH2AX、ATM和DNAPKcs共定位，并发现SEI1通过调节DNA损伤修复相关蛋白的亚细胞分布及表达，促进DNA损伤反应并出现基因组的不稳定。我们的研究还发现，SEI-1基因可以诱导鼠成纤维细胞NIH-3T3成瘤并产生DMs，且伴有ILEI（即FAM3C）基因的高扩增和过表达，而MET通路的激活起到了重要的作用。本研究揭示了SEI-1-ILEI在卵巢癌中的功能和相关机制，为肿瘤的治疗提供了分子基础。