E2/ERα通过ElncRNA2调控c-myc影响卵巢ERα(+)上皮细胞增殖的分子机制

The increasing secretion of estrogen and its combination with ERα can result in the expression of c-myc, a proliferation related gene, and the occurrence of some estrogen dependent diseases increased. However, the mechanism is still unclear. In addition to the classic estrogen-receptor signal transduction pathways, there may be some other regulation pathways exist since the c-myc gene doesn’t have the estrogen response element(ERE) .Based on our previous studies ,we believe that lncRNA plays an important role in the downstream regulation of E2/ERα and influences the biological behaviors of ERα(+)ovarian epithelial cells. Through bioinformatic analysis, we found that the predicted target gene of ElncRNA2 (Estrogen-induced lncRNA-2) was c-myc. Based on those findings ,we are trying to explore how does estrogen regulate the histone modification effect of RIZ1, a H3K9 methyltransferase, through its mediation of ElncRNA2 via ERα. What’s more, to explore its regulation mechanism of c-myc gene and its role on the proliferation ability of ERα(+) ovarian epithelial cells in further. Our research will be done by using a series assays including: competitive antagonist of E2 and ERα-shRNA, over-expression/interference technology, dual-luciferase reporter gene, RNA pulldown and mass spectral analysis, RIP and ChIP assay, and Edman sequencing technology. Through all those experiments above, we believe that our study will help interpret a new mechanism, except the classic estrogen-receptor signal transduction pathways, on E2/ERα downstream regulation from the perspective of lncRNA. It may be helpful for our better understanding of the pathogenesis of estrogen dependent ovarian diseases and can also inspire new ideas for its treatment.

E2/ERα导致c-myc基因的表达上调，使雌激素依赖性疾病的患病率增加，但机制尚不明确。由于c-myc基因启动子区没有雌激素反应原件，因此除经典的雌激素受体信号转导途径之外，可能存在其他途径调控E2/ERα下游编码基因的表达。在前期研究基础上，我们认为lncRNA是其中的关键分子，影响卵巢ERα(+)上皮细胞的生物学行为。生物信息学分析发现ElncRNA2的预测靶基因是c-myc。因此，本课题拟通过过表达/干扰技术、荧光素酶报告基因、RNA Pulldown和质谱分析、RIP实验、ChIP实验以及Edman测序技术，研究雌激素如何通过ElncRNA2调控H3K9甲基转移酶RIZ1的组蛋白修饰作用，进而调控c-myc基因的表达以及其对卵巢ERα(+)上皮细胞增殖的影响。从lncRNA调控表观遗传学的角度，为E2/ERα调控下游编码基因阐释全新机制，为雌激素依赖性卵巢疾患的治疗提供新思路。

上皮性卵巢癌（Epithelial ovarian cancer, EOC）约占所有卵巢恶性肿瘤的90%，是死亡率最高的妇科恶性肿瘤，严重威胁着妇女的生命。这一事实在很大程度上是由于该病起病隐匿，大多数患者发现时已为晚期，且常伴有广泛的腹腔转移。尽管化疗和手术治疗在不断进步，但晚期卵巢癌患者的预后仍然较差。近年来，长度大于200nt的长链非编码RNA（long non-coding RNA, lncRNA）成为肿瘤领域的明星分子，因其在肿瘤中的广泛调控作用受到越来越多的关注。许多lncRNA，诸如HOTAIR, MALAT1, ANRIL等都被证实可以作为肿瘤预后的标志物和药物治疗靶点。深入研究lncRNA在卵巢癌中的作用可以帮助我们更好的理解疾病发生发展的分子机制，并有望为改善卵巢癌患者的结局提供新的预后指标和治疗靶点。. 我们的目标lncRNA : RAD51-AS1（RAD51 antisense RNA 1，也被称为TODRA，Transcribed in the Opposite Direction of RAD51）位于15q15.1，有2个外显子，可能在卵巢癌的发生发展中发挥重要作用。本课题探索了lncRNA RAD51-AS1在上皮性卵巢癌中的表达情况、临床意义、生物学功能和作用机制。首先，我们利用荧光素酶报告和染色质免疫共沉淀实验，证实目标lncRNA是E2F1的下游靶基因，并在EOC中受负调控。然后，我们用原位杂交（ISH）检测了其在EOC中的表达。目标lncRNA定位于细胞核，是EOC患者生存率低的重要临床指标，也是EOC的独立预后因子。过表达目标lncRNA促进EOC细胞增殖，而沉默目标lncRNA抑制EOC细胞增殖，延缓细胞周期进程，促进细胞凋亡。目标lncRNA可能通过调节p53和p53相关基因参与肿瘤的发生。我们的研究明确了目标lncRNA可能成为EOC的新型预后标志物。此外，基于其对抑癌基因p53的调控， 有望为EOC的分子靶向治疗提供新思路。