HIF-lα-Notch信号通路在缺氧微环境诱导乳腺癌细胞上皮间质转化中的作用及机制研究

Despite noticeable improvement in diagnosis and treatment of breast caner, the intrinsic molecular mechanism involved in cell migration and invasion still remain unclear, and also is the key point of researches in this field. Epithelial-mesenchymal transition (EMT) are considered to have great roles on metastasis and recurrence. Hypoxia exists during the whole time of solid cancer progression and treatments. And researches on the relationship between hypoxia and cancer cells play an important role in metastasis induced by crosstalk between cancer cells and microenvironment. Hypoxia could induce EMT of cancer cells and promote metastasis by many signaling Pathways.It is unclear that the Role and mechanism of hypoxia-inducible factor-1a-Notch Signaling Pathway in EMT of human breast cancer cells under Hypoxic Conditions. In present study, human breast cancer cell lines MDA-468, MDA-231, MCF7, T47D, SK-BR-3 and were cultured under 21% O2 or 1% O2 conditions for the indicated time.The expression differences of HIF-lα, Notch-1，JAGGED1，the Notch target gene HEY1、E-cadherin, N-cadherin, Vimentin,β-catenin were tested by real-time PCR and Western blot. The capability of immigration and invasion is measured by Would healing and Matrigel transwell. Tumor formation in nude mice is evaluated. Above contents were evaluated after applying siRNA of HIF-1α, γ-Secretase inhibitors (GSI) and small interfering RNA transfection as down-regulators for Notch-1 pathway activity and NICD transfection as up-regulators,respectively. Moreover,expression of HIF-lα, Notch-1，JAGGED1，HEY1、E-cadherin, N-cadherin,Vimentin,β-catenin protein was examined by immunohistochemistry in 150 surgically resected specimens of breast cancer and adjacent tissues. The relationships between above proteins expression and clinicopathological and prognostic variables of patients were further evaluated. This project will Reveal the role and mechanism of hypoxia-inducible factor-1a-Notch Signaling Pathway in EMT of human breast cancer cells under Hypoxic Conditions.

低氧可诱导肿瘤细胞发生EMT，其调控机制涉及众多信号通路，HIF-lα-Notch通路在缺氧诱导乳腺癌细胞发生EMT中的作用尚不明确。本实验分别在常氧和缺氧条件下用RT-PCR和蛋白质印迹等方法检测6株乳腺癌细胞HIF-lα、Notch-1、JAGGED1、HEY1表达及EMT表型，用划痕和穿膜实验检测迁移侵袭能力；分别应用hiRNA干扰HIF-lα表达、γ分泌酶抑制剂阻滞Notch-信号通路、Notch-1siRNA下调Notch-通路以及转染人NICD基因质粒上调Notch-通路活性等手段，观察EMT表型、迁移侵袭能力、裸鼠内成瘤及HIF-lα-Notch通路表达；采用免疫组化法检测HIF-lα-Notch通路相关蛋白及EMT标记物在150例乳腺癌组织中的表达情况并评价其与患者临床病理以及预后指标的相关性。从而探讨IF-lα-Notch通路在缺氧诱导乳腺癌细胞发生EMT中的作用及机制

研究背景：近年来发现HIF-1a以及Notch信号通路可能影响乳腺癌发生EMT现象，但具体联系及相互机制尚不明确。.研究内容：1.研究乳腺癌细胞在缺氧条件下是否能够诱发EMT现象；2探讨Notch信号通路对乳腺癌细胞发生EMT现象的调控作用；3.检测干扰HIF-1a表达后Notch信号通路相关蛋白及EMT标志物的表达水平及细胞侵袭转移能力的变化；4.检测150例临床乳腺浸润性乳腺癌和癌旁组织标本中Notch信号通路相关蛋白以及EMT个标志物蛋白表达水平，分析其与患者临床病理特点及预后的相关性。.重要结果：1.缺氧微环境可能诱导乳腺癌细胞发生EMT现象；2. 在乳腺癌细胞发生EMT过程中Notch信号通路可能发挥了部分调控作用；3.在低氧环境中HIF-1a可能促进乳腺癌细胞发生EMT现象；4.HIF-1a可提高乳腺癌细胞的侵袭转移能力；5.乳腺癌组织中可能存在EMT现象；6. Notch信号通路可能影响HIF-1a的表达。.关键数据：1.乳腺癌MCF-7细胞在常氧和低氧环境中培养24、48h，与常氧条件相比，MCF-7细胞的E-Cadherin24h表达降低；48h表达升高；Beta –Catenin 24h、48h差异均无统计学意义;N-Cadherin24h表达升高，48h表达降低;Vimentin 24h的表达升高；差异有统计学意义(P＜0.05)。2.乳腺癌MDA-MB-231细胞常24、常24hr、常48、常48r组四组EMT标志物的表达，与常氧相比，常氧r组上皮标志物E-Cadherin24h的表达降低、48h的表达升高，差异有统计学意义（P＜0.05）；Beta –Catenin48h的表达升高；间质标志物N-Cadherin 24h的表达的降低； Vimentin的表达24h降低，48h升高，差异有统计学意义（P＜0.05）.3.150例乳腺浸润性导管癌组织中Notch-1、Jagged1及EMT标志物E-Cadherin、β-catenin、Vimentin、N-Cadherin的阳性率分别为78.7%、63.3%、58.7%、56.0%、37.3%、80.7%，癌旁组织中的阳性率为33.3%、30.0%、78.3%、75.0%、0.0%、60.0%（p＜0.05）.科学意义：本项目期望获得针对乳腺癌侵袭和转移特性的治疗靶点。