慢病毒介导ILK基因调节Wnt/β-catenin信号通路在卵巢癌上皮间质转化中的作用机制研究

Ovarian cancer occupied the first fatality rate among carcinoma in female reproductive system. Researchers showed that Integrin-Linked Kinase (ILK) posed very important role on both occurrence and development of ovarian cancer. In our preliminary experimental study, construct four plasmids system of optimized novel HIV-1 lentiviral vector successfully by segregated wide type HIV-1 genome into four plasmids, plasmid of lentivirus-mediated shRNA was established to inhibit the ILK gene, transfected ovarian cancer cell lines SKOV3 in vitro to inhibit the proliferation and improve the apoptosis of SKOV3 cells afterthat. And we also found the apoptosis approached mainly by mitochondria pathway and death receptor pathways. So we were inspired to explorer the real mechanism of ILK inhibition of proliferation of ovarian cancer cells. This project is to use lentivirus-mediated shRNA-ILK to transfect the ovarian cancer cells, SKOV3, then the regulation of proliferation, apoptosis and epithelial-mesenchymal transition (EMT) of SKOV3 by inhibition of ILK gene will be detected from mRNA and protein levels. Also, LiCl, the activator of Wnt pathway, are going to be introduced into our project here to seek the probability of ILK functions on cells through Wnt/β-catenin signaling pathway. Moreover, in vivo experiments are also planned. Ovarian cancer on nude mouse model will be set up in vivo. The changes of structures of cancer tissues will be observed by means of pathologic histology sections. The expression of β-catenin which is the key part in Wnt/β-catenin signaling pathway will be also checked by immunostaining. All these experiments will give help to uncover the pathogeny of ovarian cancer. It will lay the foundation for ovarian cancer pathogenesis and provide a new target for treatment of ovarian cancer. The clinical application in near future can also gain theoretical supports from our studies.

卵巢癌在女性生殖系统肿瘤中死亡率占首位，研究表明整合素连接激酶（ILK）在卵巢癌发生发展中起到重要作用。本课题组前期通过野生型HIV-1基因组构建优化新型慢病毒载体四质粒包装系统， shRNA-ILK转染至卵巢癌细胞SKOV3，发现抑制ILK 基因表达可抑制细胞增殖，促进凋亡，凋亡途径至少部分经由线粒体途径及死亡受体途径。本课题拟通过优化慢病毒介导的shRNA-ILK体外转染SKOV3细胞，从mRNA水平及蛋白质水平揭示如何通过抑制ILK基因调控细胞的增殖、凋亡以及上皮间质转化等病理生理过程；同时利用氯化锂激活Wnt通路，探索ILK通过Wnt/β-catenin通路调控生物功能的可能性。制作动物模型，观察肿瘤组织结构的变化及免疫组化等实验检测Wnt通路枢纽蛋白等的表达，为进一步揭示卵巢癌的可能发病机理奠定实验基础，优化基因载体系统，为治疗提供新靶点，为其临床应用提供理论支持。

卵巢癌由多基因位点共同参与的复杂疾病，涉及许多生物学过程。本课题首次研究证实整合素连接酶基因（ILK）沉默导致卵巢癌细胞的凋亡。microRNA是一类内源性非编码RNA，在各个生物学领域被广泛研究，包括复杂疾病的诊断、预防和治疗，表明miRNA在临床大范围的潜在应用前景。从miRNA表达的角度探讨ILK诱导细胞凋亡的机制。后期整合了目前已知的研究数据，运用富集分析方法发现了一些潜在的功能性miRNA-疾病相关的内在联系（MDA）。我们假设具有相似功能的miRNA更倾向于影响表型相似的疾病，对导致MDA改变的作用因子进行了数量统计分析，使用这种方法，miR-24与卵巢癌之间的一个重要的MDA被发现。相比于对照miRNA，过表达miR-24经实验验证能够显著诱发卵巢癌细胞凋亡。整合生物学数据，为功能性MDA的研究提供了新见解，建立了一个确定高可信度相关体的可行方法。