“FOX/M1miRNA/靶蛋白”网络调控卵巢癌细胞自噬的分子机制研究

As the most deadly gynecological cancer, the overall effect of treatment for ovarian cancer is still not optimistic. Autophagy is the biological processes that cells phagocytic and digest their own cytoplasm or organelles. Moderately autophagy can promote cell proliferation, but excessive self- autophagy can induce type II program cell death. Malignant tumors often in an unbalanced state of autophagy were confirmed. FOXM1 which high expressed in some tumors is a key transcription factor promoting cell proliferation. Our previous studies have shown that inhibition of FOXM1 expression can significantly increase autophagy activity and induce type II program cell death in ovarian cancer cells, but its mechanism needs to be proven. miRNAs are non-coding RNA with the regulation of gene expression in post-transcriptional level, it can regulate a variety of target genes. Ovarian cancer is the research model in this project. We intended to identify the differential expression of miRNAs in cells before and after knockdown FOXM1, at the same time applying yeast three-hybrid, bioinformatics and other methods to explore the differences in miRNA-related protein and target genes. A molecular network composed by "FOXM / miRNA / target proteins" will be formed, in which a number of new molecules involved in autophagy regulation will be found out. The function of the new molecular of autophagy will be revealed, a common regulatory pathway or cross-regulation point of proliferation and apoptosis may be proven. The objective of this research project is to provide a new theoretical basis for a better understanding of the pathogenesis of ovarian cancer in order to provide a new molecular target for its treatment.

卵巢癌是致死率最高的妇科肿瘤，其整体治疗效果差。自噬是细胞自身消化的过程，适度自噬可促进细胞增殖，过度自噬则可诱发细胞死亡；研究证实恶性肿瘤常处于自噬失衡状态。肿瘤高表达的FOXM1是促进细胞增殖的关键转录因子，我们前期研究表明，抑制FOXM1表达可显著提高卵巢癌细胞自噬活性，诱导细胞死亡而抑制细胞增殖，其机制亟待探明。miRNA是在转录后水平调控基因表达的非编码RNA，具有调控多种靶基因的特性。本项目以卵巢癌为研究模型，找出敲减细胞FOXM1前后差表达的miRNA，并应用酵母三杂交、生物信息学等方法探寻差异miRNA相关蛋白与靶基因，形成一个以"FOXM/miRNA/靶蛋白"为框架、逐步放大的分子网络，以期从中筛选出参与自噬调控的新分子，并揭示其行使自噬的分子功能，探明与增殖、凋亡等可能存在的共同调控通路或交叉调控点，以期为卵巢癌的发病机制提供新的理论依据，为其治疗提供新的分子靶标。

卵巢癌是致死率最高的妇科肿瘤，目前仍欠缺有效的治疗手段，探明卵巢发病机制，以期寻找治疗卵巢癌新的治疗方案极为重要。自噬是细胞自身消化的过程，适度自噬可促进细胞增殖，自噬与肿瘤关系密切。本研究以“FOXM1/miRNA/靶蛋白”调控卵巢癌细胞自噬的分子机制为主线展开科学研究。首先我们从3株卵巢癌细胞株中，敲除FOXM1基因后，利用miRNA芯片技术分析基因敲除前后miRNA表达差异性，并对差异显著的miRNA进行细胞及临床标本的分子生物学验证，获得了10个FOXM1相关差异miRNA，其中表达下调的有4个：miR-199a-5p、miR-1297、miR-659、miR-185，而miRPlus-E1201、miRPlus-E1074、miR-200a、miR-21、miR-155、miR-409-3p等6个miRNA表达上调。改变上述10个miRNA在卵巢癌细胞中的表达后，发现miR-199a-5p及miR-155的表达变化可影响细胞自噬活性，其中miR-199a-5p对自噬发挥负性调控作用，而miR-155发挥正性调控作用，通过生物信息学手段，我们预测并验证证实了DRAM1、Foxc2、ATG4D为miR-199a-5p的靶基因，而ULK2为miR-155的靶基因，对靶基因进行功能鉴定分析时发现，DRAM1、ATG4D、ULK2三者表达上调时细胞自噬水平明显上升，抑制mTOR信号通路也可导致3者表达上升，但其DRAM1、ATG4D、ULK2上升无法影响mTOR通路的分子表达，这一现象在卵巢癌裸鼠移植瘤上也得到了证实。因此，本研究探明了FOXM1主要通过调控miR-199a-5p及miR-155正性或负性调控其靶基因DRAM1、ATG4D、ULK2的表达而影响细胞自噬。我们的研究获得了较多与肿瘤发生关系密切的差异性miRNA，为后续深入研究奠定了基础，成功绘制“FOXM1/miRNA/靶蛋白”调控自噬的分子网络图，为认识卵巢癌发病机制提供了新的实验依据。