雌激素β受体亚型1、2、5在肺腺癌进展中的作用及机制研究

Numerous studies have comfirmed that estrogen can promote the progress of lung adenocarcinoma (LADE) through Estrogen Receptor β (ERβ), while the underlying mechanisms are poorly understood. Recent reasearches demonstrate that ERβ contains 1-5 isoforms, which have tissue specificity of the expression and function notwithstanding，there has not been any report of isoforms in LADE. Thus the study on the expression, function and mechanism of ERβ isoforms is seen as an important basis of the clarification of the issue that estrogen promotes the progress of LADE. On the basis of pre-experiment and the long-period study on the mechanism of which ERβ and Insulin-like Growth Factor-1 Receptor (IGF-1R) promote the progress of LADE, we propose several hypotheses: ERβ1,2,5 over-expressed in LADE, and they not only play different roles in LADE also regulate the signaling pathway of IGF-1R. Our project first investigate mRNA and protein expression of ERβ1,2,5, IGF-1R, MAPK, and AKT by real-time PCR and immunohistochemistry as well as Western-blot in 200 LADE tissues, and analyze association between their expression and clinicopathological factors and prognosis, to determine whether ERβ1,2,5 may play an important role in the progession of LADE. Furthmore, we will analyze the correlation of the expression of ERβ1,2,5, and IGF-1R, MAPK, and AKT to determine the molecular basis of interaction between the ERβand IGF-1R signaling pathways. We will generate stable cell lines with high- or low-expression of ERβ1,2,5 from RERF-LC-OK,A549,201T LADE cells, and examine the effects of ERβ1,2,5 on the proliferation, apoptosis, invasion, and migration after intervened by estradiol, via BrdU, AnnexinV/PI, and transwell assays, to elucidate the different roles of ERβ1,2,5 in LADE. Furthmore, to clarify whether ERβ1,2,5 may regulate the IGF-1R signaling pathway through genomic and nongenomic mechanisms, we will detect the activation of IGF-1R promoter by Dual-Luciferase Reporter and Chromatin immunoprecipitation Assays, and detect the expression of IGF-1R, MAPK, and AKT by Western-blot and real-time PCR. We will use a urethane-induced LADE model of ERβ gene knockout mice which steadily expresses ERβ1,2,5 respectively, by following inhalation of lentivirus vector expressing ERβ1,2,5, and treated with estradiol. Tumor multiplicity, tumor volume, and histological grade will be determined at 20 weeks after estradiol administration, to determine whether ERβ1,2,5 may play an important role in the genesis and development of LADE. Furthmore, we will examine the expression of ERβ1,2,5, IGF-1R, MAPK, and AKT in the mice LADE tissue, to determine whether ERβ1,2,5 may regulate the IGF-1R signaling pathway. This project contributes to both clarifying the mechanism of estrogen's promotion to the progress of lung adenocarcinoma and seeking more precise target of the targeted therapy of lung adenocarcinoma based on estrogen signal.

大量研究证实雌激素通过雌激素受体β（ERβ）促进肺腺癌进展，但机制尚不明确。ERβ有亚型1-5，研究亚型表达、作用和机制是进一步阐明雌激素促癌机制的重要基础，但在肺癌中尚无报道。我们在多年研究ERβ与胰岛素样生长因子1受体（IGF-1R）对肺癌作用机制和预实验基础上提出假说：肺腺癌过表达ERβ1、2、5，作用各不相同，且对IGF-1R信号通路有调控作用。本项目扩大样本检测肺腺癌组织中ERβ1、2、5的表达；体外实验建立稳定高或低表达ERβ1、2、5肺腺癌细胞，检测增殖、凋亡、迁移等效应和IGF-1R启动子活性及关键分子表达，探讨ERβ亚型在肺癌中的不同作用及对IGF-1R通路的影响；通过质粒构建、慢病毒感染和乌拉坦诱导建立稳定表达ERβ1、2、5的 ERβ基因敲除小鼠肺腺癌模型进一步验证上述结论。本项目对阐明雌激素促进肺腺癌进展机制及寻找基于雌激素信号的肺癌靶向治疗更精准靶点有重要作用。

项目背景：大量研究证实雌激素通过雌激素受体β（ERβ）促进肺腺癌进展，但机制尚不明确。ERβ有亚型1-5，研究亚型表达、作用和机制是进一步阐明雌激素促癌机制的重要基础，但在肺癌中相关研究尚无报道。.研究内容：我们多年研究ERβ与胰岛素样生长因子1 受体（IGF-1R）对肺癌作用机制，通过临床标本研究、体内试验、体外实验三个角度验证了不同的雌激素受体ERβ亚型在肺腺癌进展过程中存在差异性。探讨了ERβ1/2/5在肺腺癌进展众的作用及可能机制。.重要结果：.1、.首先通过对临床标本的收集与随访使用IHC方法检测发现：肺腺癌中仅ERβ1、2、5 过表达，无ERβ3、4 表达。探讨ERβ1、2、5 与肺腺癌临床病理学特征及预后的相关性,结果证实在肺腺癌过表达ERβ1、2、5，作用各不相同，且对IGF-1R信号通路有调控作用；并且在肺腺癌组织中不同程度的ERβ亚型表达是与患者预后密切相关的。.2、.体外实验建立稳定高或低表达ERβ1、2、5 肺腺癌细胞，检测增殖、凋亡、迁移等效应：具体使用A549/H1793细胞系构建siRNA-ERβ1、siRNA-ERβ5。ERβ1、5敲降后NSCLC细胞增殖、侵袭、转移能力减弱.3、.证实不同ERβ亚型和IGF-1R 启动子活性及关键分子表达相关，ERβ亚型的不同在肺癌中的不同作用及对IGF-1R 通路激活程度是不同的；.4、.通过质粒构建、慢病毒感染和乌拉坦诱导建立稳定表达ERβ1、2、5 的ERβ基因敲除小鼠肺腺癌模型进一步验证了上述结论，雌激素通过ERβ1、5促进小鼠皮下瘤生长。.科学意义：本项目对阐明雌激素促进肺腺癌进展机制，明确了雌激素受体β亚型不同对于肺腺癌进展的不同促进作用。为寻找基于抗雌激素信号的肺癌靶向治疗提供了更精准靶点。填补了在雌激素促进肺腺癌研究领域的空缺，通过本项目研究发表了一系列雌激素与肺腺癌的相关文章，进一步揭示了雌激素在肺腺癌进展过程中的重要作用。为肺癌治疗提供了潜在的治疗方式。