上皮性卵巢癌中miR-335低表达的机制及其临床相关性研究

miRNAs are genomic transcripts, whose expression levels are subject to multiple levels of regulation. Decreased miR-335, which has been reported in a variety of cancers, has an important role in ovarian cancer metastasis and prognosis. However, the molecular mechanism of its differential expression remains to be elucidated. Our preliminary study showed that miR-335 promoter region had enriched CpG islands and could be negatively regulated by Sp1, a highly expressed TF in ovarian cancer. This project will use 5 ' Race assays to identify miR-335 transcription start site (TSS), and luciferase reporter constructs containing the miR-335 promoter regions will be constructed accordingly. The correlation of miR-335 expression with Sp1 expression levels and miR-335 promoter methylation status will also be studied at cell and tissue levels. Sp1 binding sites in the miR-335 promoter will be characterized by ChIP assays. On the basis of this, we will also discuss the clinical relevance of promoter methylation status and Sp1 levels in ovarian cancer and the anti-tumor efficacy of Sp1 and methylation modulation. In summary, this project will are going to uncover the molecular mechanism of miR-335 dysregulation in ovarian cancer and provide a new theoretical basis for gene therapy in ovarian cancer.

miRNA和普通mRNA一样，均系基因组转录产物，其表达水平亦受多个层次的调控。miR-335在多种癌症中低表达，与卵巢癌转移及预后密切相关，但其差异表达的分子机制尚不清楚。本项目前期发现，miR-335启动子区存在大量CpG岛，并可受到卵巢癌中高表达的Sp1负性调控。本课题拟通过5 ' Race技术进一步明确初始miR-335转录起始点（TSS），并构建相应含miR-335启动子序列的荧光素酶报告基因，在细胞及组织水平，进一步研究Sp1表达水平以及miR-335启动子甲基化状态同miR-335表达的关系，并利用ChIP等技术明确Sp1与miR-335启动子元件的作用关系。在此基础上，我们也将讨论启动子甲基化程度及Sp1表达水平与临床预后等信息的相关性及对其干预后的抗瘤效能，最终在miR-335初始体合成前水平揭示miR-335在卵巢癌中低表达的分子机制，为卵巢癌病因治疗提供新的理论依据。

miR-335在卵巢癌中低表达，是卵巢癌复发的独立风险因素，且参与调控癌细胞的侵袭转移能力，但其低表达的分子机制尚不清楚。转录因子Sp1在卵巢癌中高表达，促进卵巢癌的增殖和转移。且Sp1可以通过调节DNA甲基转移酶的表达进而负向调节基因表达。本课题拟阐明Sp1是否通过抑制miR-335表达,促进肿瘤增殖和迁移。