缺氧微环境下CaSR调控子宫内膜癌上皮间质转化的机制研究

发布时间：2018-03-22 21:09

本文选题：子宫内膜癌　切入点：上皮间质转化　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：[目的]肌层浸润及远处侵袭转移是影响子宫内膜癌患者预后的主要因素。近年来越来越多的研究表明,EMT在子宫内膜癌的原位侵袭及远处转移中发挥着重要的作用,严重影响患者的预后。研究还发现,缺氧可以诱导EMT过程,同时钙敏感受体CaSR在其中可能发挥着关键作用。因此,我们探讨缺氧与子宫内膜癌EMT之间的联系,并挖掘CaSR在其中扮演的角色,阐明子宫内膜癌EMT的发生及其有关的调节因子,为后续如何阻断这一机制的发生和寻找抑制子宫内膜癌复发转移的方法提供新思路。[方法]1.IHC法检测正常和子宫内膜癌组织中HIF-1α和EMT标志分子E-cadherin、 vimentin的表达水平；2.IF法观察缺氧前后子宫内膜癌细胞系Ishikawa、Kle的细胞骨架及E-cadherin、vimentin的定位和表达水平：3.瞬时转染干扰HIF-1 α表达;4. Transwell模型观察缺氧或质粒转染细胞后其迁移和侵袭能力;5. RT-PCR和Western blotting检测缺氧或转染后细胞HIF-1α、E-cadherin、 vimentin、Snail和CaSR转录表达变化；6.构建慢病毒转染细胞沉默及过表达CaSR,荧光染料Fluo-2/AM示踪监测细胞内钙离子变化以验证CaSR表达量改变后的功能变化;RT-PCR、Western blotting和IF法分析转染前后细胞EMT相关蛋白变化及功能变化;7.CCK-8法检测细胞凋亡。1.与正常之宫内膜组织相比较,子宫内膜癌中HIF-1α和vimentin表达增高(P0.05),而E-cadherin降低,但无统计学意义(P0.05);E-cadherin表达与HIF-1α、vimentin呈负相关(P0.05),提示缺氧可能与EMT具有相关性。2.缺氧后细胞骨架重建,细胞之间连接变稀疏,间隙增宽,部分细胞伸出伪足,运动能力增强,向梭形样改变。同时E-cadherin表达降低,vimenti n表达增高,结果显示缺氧后可以促进子宫内膜癌细胞呈EMT表型变化。3.缺氧后HIF-1α、EMT相关分子vimentin、Snail转录表达增高(P0.05),而E-cadherin转录表达降低,CaSR的转录表达也明显增高(P0.01),可见缺氧后子宫内膜癌发生EMT改变,并伴随着CaSR表达的增加。4.缺氧后HIF-1α表达增加,质粒转染可以成功干扰HIF-1α表达;HIF-1α的增加可以促进子宫内膜癌EMT改变,但沉默HIF-1α表达后可以抑制缺氧引起的EMT表型产生,同时伴有肿瘤细胞侵袭能力的减弱(P0.05),但受到CaSR的负性调控。5.沉默及过表达子宫内膜癌细胞CaSR后细胞内钙离子内流分别相应的减少、增加;沉默CaSR表达促进细胞增殖,过表达CaSR抑制细胞的增殖；CaSR可以抑制H IF-1α表达,同时抑制EMT形成(E-cadherin增高及Vimentin降低),其伴随着β-catenin的表达增加及定位的改变。[结论]缺氧后子宫内膜癌可以发生EMT,其通路至少部分依赖HIF-1 α的增加,但受CaSR负性调节。缺氧还可以促进CaSR表达,进而抑制子宫内膜癌细胞的增殖及改变β-catenin胞膜／胞浆定位,参与抑制EMT发生,但其内在机制还有待进一步深究,以为干扰子宫内膜癌EMT调控提供更可靠的理论基础。
[Abstract]:[objective] Myometrium infiltration and distant invasion and metastasis are the main factors influencing the prognosis of endometrial carcinoma. In recent years, more and more studies have shown that EMT plays an important role in the in situ invasion and distant metastasis of endometrial carcinoma. The study also found that hypoxia can induce the process of EMT, in which calcium sensitive receptor CaSR may play a key role. Therefore, we explore the relationship between hypoxia and EMT in endometrial carcinoma. And excavate the role of CaSR in it to elucidate the occurrence of EMT in endometrial carcinoma and its related regulatory factors. To provide a new way to block the occurrence of this mechanism and to find a way to inhibit the recurrence and metastasis of endometrial carcinoma. [methods] 1.IHC method was used to detect the expression of E-cadherin and vimentin in normal and endometrial carcinoma tissues. The cytoskeleton of endometrial carcinoma cell line Ishikawaa Kle and the localization and expression level of E-cadherin vimentin were observed before and after hypoxia. Transient transfection interfered with the expression of HIF-1 伪. The Transwell model was used to observe the ability of migration and invasion after hypoxia or plasmid transfection. 5. RT-PCR and Western blotting. To detect the transcriptional changes of HIF-1 伪伪 -E-cadherin, vimentininSnail and CaSR after hypoxia or transfection. To construct lentivirus-transfected cells for silencing and overexpression of CaSR, fluorescent dye Fluo-2/AM tracer was used to monitor the changes of intracellular calcium in order to verify the functional changes of RT-PCRG after the changes of CaSR expression. Blotting and if methods were used to analyze the changes of EMT related proteins and their functions before and after transfection. The apoptosis of cells was detected by CCK-8 method and compared with normal endometrial tissue. In endometrial carcinoma, the expression of HIF-1 伪 and vimentin increased and E-cadherin decreased, but the expression of E-cadherin was negatively correlated with HIF-1 伪 vimentin, suggesting that hypoxia may have a correlation with EMT. 2. After hypoxia, the cytoskeleton was reconstructed, the connections between cells became sparse, and the gap widened. Some of the cells extended pseudopodia, increased their motor ability and changed to spindle shape. Meanwhile, the expression of E-cadherin decreased, and the expression of vimenti n increased. The results showed that hypoxia could promote the expression of EMT phenotypic changes in endometrial carcinoma cells. After hypoxia, the expression of HIF-1 伪 -EMT-related molecule vimentininSnail was increased (P0.05A), while the expression of E-cadherin decreased significantly (P 0.01). The EMT changes in endometrial carcinoma after hypoxia were observed. With the increase of CaSR expression, the expression of HIF-1 伪 was increased after hypoxia. Plasmid transfection could successfully interfere with the expression of HIF-1 伪 and increase the expression of HIF-1 伪, but silencing the expression of HIF-1 伪 could inhibit the phenotypic production of EMT induced by hypoxia. At the same time, the invasion ability of tumor cells was weakened (P 0.05), but it was regulated negatively by CaSR. 5. The intracellular calcium influx decreased and increased after silencing and overexpression of endometrial cancer cells CaSR, and the silencing of CaSR expression promoted cell proliferation. Overexpression of CaSR inhibits cell proliferation and CaSR can inhibit the expression of H IF-1 伪. At the same time, the increase of E-cadherin and the decrease of Vimentin were inhibited by EMT, which was accompanied by the increase of 尾 -catenin expression and the change of localization. [conclusion] EMTs may occur in endometrial carcinoma after hypoxia, and its pathway depends at least in part on the increase of HIF-1 伪. However, under the negative regulation of CaSR, hypoxia can also promote the expression of CaSR, thus inhibit the proliferation of endometrial cancer cells and change the localization of 尾 -catenin membrane / cytoplasm, and participate in the inhibition of EMT, but the underlying mechanism remains to be further studied. To provide a more reliable theoretical basis for interfering with EMT regulation of endometrial carcinoma.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.33

【相似文献】