HMGA2介导FSH促进输卵管伞端上皮发生EMT的机制研究

发布时间：2018-01-26 03:12

本文关键词： 卵巢高级别浆液性腺癌卵泡刺激素 let-7b 高迁移率族蛋白A2 上皮间质转化　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：目前有学者认为,部分卵巢高级别浆液性腺癌(high grade serous cancer,HGSC)可能起源于输卵管伞端上皮细胞(主要是输卵管上皮分泌细胞),在各种因子或环境等的刺激下出现“P53印迹”,随后出现输卵管上皮内癌(serous tubal intraepithelial carcinoma, STIC),最后成为侵袭性的浆液性腺癌[1,2]。上皮细胞-间充质细胞转化(epithelial-mesenchymal transitions,EMT)指上皮细胞在形态学上发生成纤维细胞或间充质细胞表型的转变并获得迁移的能力,与组织重建再生、肿瘤的发生发展、转移密切相关。有研究表明,正常上皮细胞在各种因素作用下可发生EMT,参与致瘤[3]。高迁移率蛋白A2 (High mobility group proteinA2, HMGA2)是HMGA家族成员之一,其表达水平与肿瘤的恶性程度、转移机率及预后密切相关[4]。目前国内外很多研究都集中在"P53印迹”出现之后,尤其是STIC,但对“P53印迹”出现之前细胞的变化研究较少,而研究该阶段出现的事件能为掌握HGSC的发生机制、研究早期诊断及早期靶向干预奠定基础。籍于此,本研究探讨了FSH作用于FTE后细胞是否发生EMT,且探究HMGA2在此过程中发挥的作用。第一部分HGSC和LGSC肿瘤未累及输卵管伞端的鉴别目的：了解在P53突变之前,HGSC中未累及的输卵管伞上皮细胞的某些指标是否已经发生了变化。方法：方法选择输卵管未累及的卵巢上皮高级别浆液性腺癌28例,低级别浆液性腺癌24例,另取因盆底功能障碍而切除全子宫双附件5例做对照。选取输卵管伞端组织行免疫组化检测。基因突变指标：P53；增殖指标：pAKT;血管内皮生长因子：VEGF;转移指标：E-cadherin;侵袭指标：MMP-2;肿瘤微环境指标：COX-2。结果：高级别浆液性腺癌伞端的pAKT和COX-2表达明显高于低级别浆液性腺癌(分别为61% vs 8%和71 %vs 21%,P=0.005和0.007,).低级别浆液性腺癌伞端E-Cadherin的表达明显高于高级别浆液性腺癌(83% vs 21%,P=0.003)。 MMP-2, VEGF和p53在高、低级别浆液性腺癌中的表达差异无统计学意义(分别为21% vs 13%,25% vs 21%和14% vs 8%,P=0.78,0.86和0.82)。结论：高级别浆液性卵巢腺癌中未受肿瘤累及的伞端细胞在p53突变发生前其增生、粘附力及炎症微环境就可能已经发生了变化。第二部分卵泡刺激素FSHHGSC伞端上皮细胞发生EMT目的：了解FSH能否促进HGSC伞端上皮细胞发生EMT方法：取前期已成功原代培养的低级别浆液性腺癌、高级别浆液性腺癌肿瘤未累及、P53(-)的输卵管伞端上皮细胞。将FSH以不同浓度(0、10、20、40、 80、160mIU/mL)和不同时间(分别作用于0、12、24、48、72h)作用于FTE,观察细胞形态变化情况。Western-blot检测EMT标志物E-Cadherirn、 N-Cadheriin、波形蛋白(Vimentin)、MMP-2及P53蛋白表达情况。RT-PCR检测mRNA表达情况。结果：FSH浓度为40mIU/ml作用于HGSC的FTE细胞48h后,上皮细胞外形演变为纺锤形纤维细胞形态,EMT标志物E-Cadherin明显下调,而N-Cadherin、Vimentin、MMP-2明显上调,且呈FSH浓度-作用时间依赖性,初步证实了发生EMT。而在LGSC未见这些变化。FSH的作用不能促进P53蛋白表达改变。结论：FSH促进了HGSC伞端上皮细胞发生EMT。第三部分：HMGA2介导FSHHGSC输卵管伞端上皮发生EMT目的：了解]HMGA2在FSH促进HGSC输卵管伞端上皮发生EMT中的作用。方法：取前期已成功原代培养的HGSC中瘤未累及、P53(-)的输卵管伞端上皮细胞。将FSH以不同浓度(0、0、20、40、80mIU/mL)和不同时间(分别作用于0、12、24、48、72h)作用于FTE。 Western-blot检测HMGA2蛋白表达情况。RT-PCR检测let-7b mRNA表达情况。FTE细胞转染]HMGA2 shRNA后,浓度为40mIU/mL的FSH作用,观察细胞是否发生EMT及EMT相关分子E-cadherin, MMP-2, N-cadherin,vimentin蛋白表达变化情况。观察分子生物学方法敲除let-7b后,HMGA2蛋白表达情况。结果：FSH作用HGSC的FTE细胞后,HMGA2蛋白表达逐渐增加,let-7b mRNA表达逐渐降低,并呈浓度-时间依赖性。FTE细胞转染HMGA2 shRNA后,FSH不能促进FTE细胞发生EMT,且EMT相关分子E-cadherin, MMP-2, N-cadherin,vimentin蛋白表达量均未出现明显变化。Anti-let-7b转染FTE细胞后,HMGA2蛋白表达升高。结论:HMGA2在介导FSH促进FTE发生EMT中起了重要作用。let-7b能够调控HMGA2的表达。综上所述,FSH促进了HGSC伞端上皮细胞发生EMT,且最佳条件为浓度40mIU/ml、作用时间48h。 FSH能调控FTE中let-7b的下调并通过其而上调HMGA2的表达。HMGA2介导FSH促进HGSC的FTE细胞发生EMT,而在LGSC中未发现这种现象。本课题的研究结果提示了FSH通过HMGA2在人类高级别卵巢浆液性腺癌中具有促进肿瘤进展的作用,并初步探索了相关的分子调节机制,为进一步阐明高级别浆液性卵巢腺癌发生、发展的机制提供了新的思路,并为其的诊断、治疗提供了可能的新线索。
[Abstract]:At present, some scholars believe that some high grade ovarian serousadenocarcinoma (high grade serous cancer, HGSC) may originate from fallopian tube epithelial cells (mainly secretory cells of the oviduct), in a variety of factors or environmental stimuli "P53 blot, followed by tubal intraepithelial carcinoma (serous tubal intraepithelial carcinoma, STIC), and finally become invasive serous carcinoma [1,2]. epithelial mesenchymal transformation (epithelial-mesenchymal transitions EMT) refers to the transformation of epithelial cells of fiber cells or mesenchymal cell phenotype in morphology and migration ability of regeneration and tissue reconstruction, the occurrence and development of tumor. Metastasis. Studies have shown that normal epithelial cells occurred in EMT under the influence of various factors, involved in the tumorigenic [3]. high mobility protein A2 (High mobility group proteinA2, HMGA2) Is a member of the HMGA family, its expression and tumor malignant degree, metastasis and prognosis of [4]. probability at home and abroad, many studies have focused on the "P53 mark", especially the STIC, but the P53 imprinting changes of cells before, and on the stage of the event to to grasp the mechanism of HGSC on the early diagnosis and targeted intervention to lay the foundation. Base on this, this study investigated the effect of FSH on whether FTE cells occurred after the EMT, and explore the play the role of HMGA2 in this process. The first part of the HGSC and LGSC to identify tumor uninvolved fallopian tube: understanding in P53 the mutation before, some indicators did not involve HGSC in fallopian tube epithelial cells have changed. Methods: select 28 cases of tubal ovarian high grade serous adenocarcinoma without involvement of the lower level 24 cases of serous adenocarcinoma, another for pelvic floor dysfunction and resection of the uterus double Appendix 5 cases in the control. Select the fallopian tube tissue by immunohistochemistry. Gene mutation index: P53; proliferation index: pAKT; vascular endothelial growth factor VEGF; transfer index: E-cadherin; MMP-2; tumor invasion index micro environmental indicators: COX-2. results: high grade serous adenocarcinoma of the fimbria and pAKT expression of COX-2 was significantly higher than that of low-grade serous carcinoma (61% vs 8% and 71%vs 21%, P=0.005 and 0.007). The expression of low grade serous adenocarcinoma fimbria E-Cadherin were significantly higher than those of high grade serous adenocarcinoma (83% vs 21% P=0.003). MMP-2, VEGF, and p53 in high, no statistically significant differences in the expression of low grade serous adenocarcinoma (21% vs 13%, 25% vs 21% and 14% vs 8%, P=0.78,0.86 and 0.82). Conclusion: swollen without high-grade serous ovarian adenocarcinoma Umbrella end cell tumor involved in p53 mutation occurred before the proliferation, adhesion and inflammatory microenvironment may have changed. The second part EMT to FSHHGSC follicle stimulating epithelial cells: understanding FSH fimbria can promote HGSC fimbria epithelial cells. EMT methods: Previous studies have successfully cultured lower level serous adenocarcinoma and high-grade serous ovarian cancer without involvement, P53 (-) of the fallopian tube epithelial cells. FSH at different concentrations (0,10,20,40, 80160mIU/mL) and different time (respectively for 0,12,24,48,72h) to FTE, observe the cell morphology changes of.Western-blot detection of EMT markers E-Cadherirn, N-Cadheriin, vimentin (Vimentin), the expression of MMP-2 and P53 protein expression in.RT-PCR mRNA assay. Results: the concentration of FSH is FTE 48h 40mIU/ml in HGSC cells, epithelial cells were spindle shape evolution Form of fiber cells, EMT marker E-Cadherin was down regulated, while N-Cadherin, Vimentin, MMP-2 and FSH were significantly increased, the concentration time dependence, preliminary confirmed the occurrence of EMT. in LGSC was.FSH these changes cannot promote the expression of P53 protein. Conclusion: FSH promotes HGSC fimbria epithelial cells EMT. the third part: HMGA2 FSHHGSC mediated by fallopian tube epithelial EMT Objective: to understand the]HMGA2 FSH HGSC in promoting fallopian tube epithelial EMT in vitro. Methods: Previous studies have successfully cultured HGSC tumor uninvolved, P53 (-) of the fallopian tube epithelial cells with FSH. Different concentration (0,0,20,40,80mIU/mL) and different time (respectively for 0,12,24,48,72h) to detect HMGA2 protein expression of.RT-PCR FTE. Western-blot mRNA to detect let-7b expression of.FTE cells transfected with]HMGA2 after shRNA, the concentration of FSH 40mIU/mL, EMT and EMT were observed whether related molecules E-cadherin, MMP-2, N-cadherin, vimentin protein expression changes. To observe the molecular biological methods let-7b knockdown the expression of HMGA2 protein. Results: the FSH function of HGSC FTE cells, HMGA2 protein expression increased, let-7b expression of mRNA decreased gradually, and a concentration time dependent.FTE cells transfected with HMGA2 shRNA, FSH can promote FTE cell EMT, EMT and MMP-2, related molecular E-cadherin, N-cadherin, vimentin protein expression showed no obvious change in.Anti-let-7b after transfection into FTE cells, the expression of HMGA2 protein increased. Conclusion: HMGA2 mediated FSH promote EMT play the important role of.Let-7b can regulate the expression of HMGA2 FTE. In summary, FSH promoted HGSC fimbria epithelial cells EMT, and the best conditions for the concentration of 40mIU/ml, reaction time 48h. FSH The down-regulation of let-7b in regulation of FTE and the expression of.HMGA2 mediated upregulation of HMGA2 by FSH HGSC FTE promote EMT cells, and this phenomenon was not found in LGSC. The results of this study suggest that FSH can promote tumor progression in human ovarian serous adenocarcinoma in high level by HMGA2, and preliminary exploration of the regulatory mechanism of related molecules, in order to further clarify the high-grade serous ovarian adenocarcinoma, provides new ideas for the development of the mechanism, and its diagnosis, treatment may provide a new clue.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.31

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