基于“肺肾相关”从性激素及其受体和EGFR交叉信号通路探讨肾虚内环境对非小细胞肺癌的影响及机制

Firstly, this study compared the differences between transplanted Lewis lung tumors and metastases in castrated mice of deficiency of kidney and normal mice in order to investigate whether environment of deficiency of kidney could influence on the formation of lung cancer . secondly four diagnostic methods and questionnaire was used to explore the extent , the types , clinical characteristics(TNM staging, histological type, degree of differentiation) and prognosis of NSCLC of deficiency of kidney. Thirdly, with sex hormone , its receptors as well as EGFR pathway as a starting point, RT-PCR，Western blot and other experimental techniques were used to observe the effects and gender differences that different levels of E2, T affected on xenografted tumor of Lewis lung carcinoma and metastatic tumor; to discuss that E2, T regulated expression of its receptors ERα, ERβ, AR; to analysis the correlation between E2, T ,its receptor, EGFR and downstream signal PI3K-AKT.Finally RNA interference techniques were used to specifically inhibit the expression of ER and AR of lung adenocarcinoma A549 cells ,then characteristics change of the invasion and metastasis of cancer cells were observed ,expression of EGFR and related signaling molecules were detected with real time-PCR, Western blot. The study initially revealed the mechanisms that the environment of deficiency of kidney worked on lung cancer, which also provided the basis for the hypothesis "the environment of deficiency of kidney is one of the internal reasons of development of lung cancer", from the "kidney" Treating lung cancer as well as targeted drug development and application.

本研究首先比较去势肾虚小鼠和正常小鼠Lewis肺癌移植瘤和转移瘤的差异探讨肾虚内环境对肺癌形成有无影响；其次用四诊和调查问卷的方法探讨NSCLC患者肾虚程度、肾虚类型与肺癌临床特征和预后的关系；第三，以性激素及其受体和EGFR交叉通路为切入点，采用RT-PCR、Western blot等实验技术，观察不同水平E2、T对小鼠Lewis肺癌移植瘤和转移瘤的影响及性别差异，探讨E2、T对其受体ERα、ERβ、AR表达的调控，分析E2、T与其受体、EGFR及下游信号PI3K-AKT等的相关性；利用RNA干扰技术分别特异性抑制肺腺癌A549细胞中ER和AR的表达，观察癌细胞增殖侵袭和转移的变化，用real time-PCR、等检测EGFR及相关信号分子的表达；初步揭示肾虚内环境影响肺癌的机制，为"肾虚内环境是肺癌发生发展的内因之一"的假说和从""肾"论治肺癌及靶向药物的研发和应用提供依据。

本研究首先通过去势建立了小鼠肾虚Lewis肺癌病证结合模型，肾虚组小鼠Lewis肺癌移植瘤和肺转移灶大小及数目均大(多)于正常组；其次，260例NSCLC患者中符合肾虚诊断标准者235例，重度肾虚67例，中度肾虚121例，轻度肾虚47例，多为肾阴阳两虚，肾虚比例和程度有随临床分期进展而增加的趋势，Ⅳ期110例全部肾虚，低分化者肾虚更重；病理类型与肾虚关系不大；骨转移71例中肾虚69例，脑转移27中肾虚26例，骨脑转移34例均肾虚，均为阴阳两虚以肾阴虚为主；胸腹水78例全部肾虚，多为肾阴阳两虚以肾阳虚为主，肾虚程度与疾病进展有一定关系；第三，去势肾虚Lewis肺癌小鼠和NSCLC患者性激素及其受体表达异常，去势肾虚组Lewis肺癌小鼠血清E2、T和移植瘤ERα、ERβ、AR、EGFR、PI3K、AKT的表达水平与正常组Lewis肺癌小鼠比较存在差异，且有性别差异；第四，siRNA特异性抑制肺癌A549细胞ERα、ERβ和AR的表达，并用不同浓度(10µmol/L、50µmol/L、100µmol/L)的雌二醇、睾酮干预A549细胞， siRNA特异性抑制AR、ERα、ERβ表达后，Western blot、Real-time PCR检测显示EGFR、PI3K、AKT蛋白和mRNA表达水平均降低，CCK-8和流式细胞术显示A549细胞活性降低、凋亡增加，划痕和Transwell实验表明A549细胞的迁移侵袭能力降低；50µmol/L T/E2干预A549细胞后，AR、ERα、ERβ表达降低，EGFR、PI3K、AKT表达也降低，A549细胞活性及迁移侵袭能力均降低、凋亡增加；10µmol/L和100µmol/L T/E2干预A549细胞后，AR、ERα、ERβ表达均升高，EGFR、PI3K、AKT表达也升高，A549细胞活性及迁移侵袭能力增强、凋亡减少；提示正常水平的性激素及其受体抑制A549细胞增殖，过低或过高水平的性激素及其受体的异常表达可通过EGFR-PI3K-AKT信号通路促进A549细胞的增殖。为“肾虚内环境是肺癌发生发展的内因之一”的假说和从肾论治肺癌及靶向药物研发和应用提供了依据。