TGFβ/ZEB/miR-200信号环路在子宫内膜癌上皮-间质转化过程中的调控作用

The TGFβ/ZEB/miR-200 signaling loop has been shown to participate in epithelial- mesenchymal transition(EMT), but further study is needed to confirm its role in endometrial carcinoma(EnCa). Previously we found that TGFβR/Smads pathway is defected in tumor stroma in EnCa and paracine factors from normal stroma cells can inhibit proliferation of EnCa cells. The aim of this study is to determine the roles of TGFβ/ZEB/miR-200 signaling loop in the process of EMT through focusing on the epithelial-stroma cross talk and the role of cancer cells that have undergone EMT in EnCa. We will study the expression and correlation of TGFβ/ZEB/miR-200 in human EnCa tissues.We will study whether reciprocal activation of cancer cells and stroma cells can stimulate EMT through TGFβ/ZEB/miR-200 signaling loop by constructing co-culture systems and try to demonstrate the role of ZEB/miR-200 double negative loop in EMT of EnCa. We will try to construct a stable mesenchymal state of endometrial cell line by transwell selection and prolonged TGF-β treatment and explore its role in EMT of EnCa through co-culture system and in changing the expression of ZEB/miR-200.Several xenograft models of different EnCa cells in nude mice will be established to confirm the role of TGFβ/ZEB/miR-200 in EMT. This study will help to find the key metastatic process in endometrial carcinoma tumor microenvironment and also help to discover new promising therapeutic approach for the treatment of endometrial carcinoma.

TGFβ/ZEB/miR-200信号环路与上皮-间质转化(EMT)有关，但在子宫内膜癌（EnCa）中缺乏深入研究。申请者研究显示，EnCa间质细胞较正常间质细胞TGFβR/Smads信号缺陷，正常间质通过旁分泌抑制EnCa细胞生长。本研究拟从上皮间质相互作用、上皮间质转化细胞两个角度，探讨该信号环路在EnCa EMT中的调控作用：研究TGFβ/ZEB/miR-200在人EnCa组织表达及相关性；建立共培养体系，研究EnCa上皮间质相互激活通过该环路对EMT的作用，证明ZEB/miR-200调控EnCa EMT；采用侵袭小室筛选+TGFβ刺激法建立EnCa上皮-间质稳定转化细胞系，与癌细胞共培养研究转化细胞对EnCa EMT的作用及ZEB/miR-200表达改变；④建立EnCa裸鼠移植瘤模型，体外验证。本研究有利于发现EnCa肿瘤微环境中转移机制的关键环节，为治疗提供新靶点。

子宫内膜癌（endometrial carcinoma ,EnCa）是女性生殖系统三大恶性肿瘤之一。上皮-间质转化（epithelial to mesenchymal transition，EMT)是肿瘤转移的关键性事件。肿瘤微环境、上皮-间质相互激活机制可促进EMT。转化生长因子β1（TGFβ1）在其中发挥重要作用。围绕本课题进行研究并得出了相应的结论：1.miR200a /ZEB1(锌指E-盒结合同源异形盒1)信号通路在EMT过程中发挥作用：在EnCa ，miR200a 与E-cadherin之间存在数量上的关系，miR200a可通过对ZEB1的调控来影响其下游靶点E-cadherin 的表达。2.EnCa肿瘤微环境及TGFβ1对NF（子宫内膜间质细胞）活化为CAF（肿瘤间质细胞）的影响：TGFβ1和CM可以促进NF向CAF的活化。3.子宫腺肌病中EMT介导Notch1/numb/snail信号通路的异常表达：Notch1/Snail/Numb信号通路可能通过影响EMT参与子宫腺肌病的发生和发展。4 .二甲双胍通过βKlotho介导的ERK1/2信号和AMPKα信号抑制17β-雌二醇诱导的EnCa细胞上皮间质转化：二甲双胍抑制17β-雌二醇诱导的EnCa的细胞增殖、迁移和侵袭并对抗17β-雌二醇的抗细胞凋亡作用。二甲双胍通过βKlotho, ERK1/2和AMPKα信号通路逆转EnCa的EMT；EnCaβKlotho的表达降低，βKlotho表达通过ERK1/2信号通路抑制17β-雌二醇诱导的EnCa细胞的增殖和EMT，与二甲双胍在EnCa细胞增殖和EMT中表现为协同作用； 二甲双胍通过刺激AMPKα信号通路抑制EnCa的EMT。5正常子宫内膜间质细胞在体外通过Slug和E-cadherin调节17β-雌二醇诱导的EnCa上皮-间质转化。6.H19/let-7轴调节内膜癌等肿瘤细胞的迁移侵袭和上皮-间质转化。7.米非司酮通过调控Ｈ19 基因甲基化抑制EnCa细胞的迁移。在本课题的基础上又做了一定扩展性的研究：Rac1/β-Catenin 信号通路通过作用于Snail和MMP9协助滋养细胞的侵袭；合并妊娠期高血压病患者胎盘中miRNA-1233的表达及Hoxb3表达的抑制，有助于研究相关疾病的发病机制。通过上述研究，进一步探索其侵袭转移机制，有利于寻找新的治疗靶点