Dicer-miR-101-EZH2正反馈通路在上皮性卵巢癌恶性进展中的作用研究

The enhancer of zeste homolog 2 (EZH2), which acts as a histone methyltransferase, contributes to epigenetic silencing of cancer suppressor genes. Our early studies and foreign researchers all showed that high levels of EZH2 expression are associated with the malignant progression of ovarian cancer. Our pioneer experiments indicated reduced miR-101 expression resulted in EZH2 over-expression in ovarian cancer. Moreover, DNA hypermethylation of the Dicer gene promoters and low expression level of Dicer are detected in ovarian cancer tissues; and depletion of EZH2 could reduces DNA hypermethylation of the Dicer gene promoters and induce Dicer expression. Based on these evidence, we raise a hypothesis that a Dicer-miR-101-EZH2 positive feedback pathway exists in ovarian cancer which promotes the malignant transformation of ovarian epithelium. To verify this hypothesis, several methods, such as luciferase assay, immunological co-precipitation and inhibition of histon methylation, will be used for clarifying the regulation mechanisms between Dicer, miR-101 and EZH2; epithelial-mesochymal transformation (EMT), cell proliferation, invasion, migration and angiogenesis will be examined after establishing this feedback pathway in normal ovarian epithelium; the effectiveness of therapy targeting Dicer-miR-101-EZH2 pathway will be showed in ovarian cancer cell lines models.

EZH2可以通过对组蛋白甲基化修饰发挥沉默抑癌基因作用，目前已知EZH2过度表达与卵巢癌恶性进展密切相关。我们和他人前期研究已经证实：调控小分子RNA成熟的关键酶Dicer在卵巢癌组织中低表达，使miR-101出现成熟障碍，而miR-101对EZH2有靶向抑制作用，一旦缺失就可产生过量EZH2。进一步研究提示：卵巢癌中Dicer基因启动子区DNA高甲基化，导致 Dicer表达低于正常组织；细胞实验降低EZH2表达， Dicer启动子区甲基化水平降低，Dicer表达上升。由此我们推测卵巢癌进展可能存在EZH2→Dicer→miR-101→EZH2正反馈通路，它可能是EZH2实现自身表达调节，推动卵巢癌恶性进展的重要机制。本课题拟在卵巢癌组织及细胞中验证此通路的完整性并阐明其机制；加强和阻断此循环通路后检测细胞EMT、增殖、侵袭及血管形成等恶性行为影响，验证该通路作为卵巢癌治疗靶点的有效性。

组蛋白甲基转移酶EZH2在上皮性卵巢癌中高表达，与卵巢癌恶性进展密切相关，但维持其高表达的机制尚不清楚。申请者前期研究发现:上皮性卵巢癌中，miR-101能抑制EZH2的表达。本课题意在研究EZH2在上皮性卵巢中高表达的机制。首先筛选出肿瘤中低表达，负向调控EZH2表达的miRNAs:hsa-mir-101-3p、let-7e、hsa-mir-26a-5p、hsa-mir-98-5p、hsa-mir-141-3p,并将上述5个miRNAs命名为Et-miRNAs(EZH2-targeted miRNAs)。发现上皮性卵巢癌中，Et-miRNAs可负向调控EZH2的表达，并影响细胞增殖能力，而在实验室前期构建的具有部分细胞干性的SKOV3 3rd中存在着相似的结果。同时发现与亲本相比，高表达EZH2的SKOV3 3rd具有低的Et-miRNAs水平，EZH2可通过PRC2途径促进Et-miRNAs启动子区甲基化调控 Et-miRNAs的表达。因此，形成EZH2/Et-miRNAs正反馈维持EZH2的高表达，促进肿瘤细胞的增殖，此发现为卵巢癌的治疗提供了新的靶点与思路。