MiR-200c通过靶向ZNF217与ZEB1抑制TGF-β信号并调控乳腺癌trastuzumab耐药与转移

发布时间：2018-04-13 18:32

  本文选题：miR-200c + TGF-β信号　； 参考：《第四军医大学》2015年博士论文

【摘要】：【背景】目前,乳腺癌已经成为最为主要的威胁女性生命健康的恶性疾病之一,该病防治是肿瘤研究领域的热点问题之一。人表皮生长因子受体2(human epidermalgrowth factor receptor-2,HER2)过表达于20%~25%乳腺癌,患者治疗预后差,易发生转移,HER2已经成为治疗乳腺癌的重要靶点。曲妥珠单抗(trastuzumab,商品名:herceptin,赫赛汀)是靶向HER2的人源化单克隆抗体,在HER2阳性乳腺癌早期治疗及转移治疗中取得较大成功。然而,trastuzumab耐药以及由此而导致的潜在转移性增加却一直困扰临床医生。近年来,乳腺癌trastuzumab耐药与乳腺癌转移研究取得显著进步,然而,乳腺癌trastuzumab耐药与转移相关性及其具体的分子机制尚需进一步阐明。TGF-β信号在肿瘤发生发展中具有重要作用并扮演着双重角色,一方面,其在肿瘤早期发挥增殖抑制与促凋亡作用,另一方面,其在肿瘤晚期可有效诱导肿瘤细胞发生并维持上皮间质转化(epithelial-mesenchymal transition,EMT),从而促进肿瘤侵袭与转移。此外,TGF-β信号在多种肿瘤放射与药物治疗抵抗中的作用逐渐引起重视,成为肿瘤研究领域的热点。越来越多的证据表明,TGF-β信号在肿瘤耐药与转移中具有重要作用,有望成为重要的肿瘤治疗靶点。然而,关于TGF-β信号在乳腺癌trastuzumab耐药及伴随潜在转移性增加中的作用及其自身调控机制尚不明确。Mi RNAs是一类小分子RNA,通过靶向调控蛋白质而广泛参与肿瘤发生、发展。因此,阐明mi RNA在介导TGF-β信号调控HER2阳性乳腺癌trastuzumab耐药和转移中的潜在作用及其分子机制,将为乳腺癌多重恶性表型的逆转提供新的思路,对于乳腺癌临床治疗具有重要意义。【目的】阐明mi RNA参与TGF-β信号调节并调控乳腺癌trastuzumab耐药与转移的潜在作用机制,为trastuzumab耐药或者转移乳腺癌治疗提供新的思路。【方法】通过持续加药培养野生型(wide type,WT)乳腺癌细胞的方法建立体外乳腺癌trastuzumab耐药(trastuzumab resistant,TR)细胞模型,MTT检测分析乳腺癌WT与TR乳腺癌细胞对trastuzumab药物敏感性。软琼脂克隆形成实验分析WT与TR乳腺癌细胞非锚定依赖生长能力;transwell侵袭实验分析WT与TR乳腺癌细胞侵袭能力;对WT与TR乳腺癌细胞进行形态学观察,实时定量PCR(quantitative realtime PCR,q PCR)检测EMT转录因子水平,WB检测EMT标记分子,以观察细胞诱发EMT。WB检测乳腺癌WT与TR乳腺癌细胞TGF-β信号激活状态,q PCR与ELISA检测分析TGF-β的表达。RNA干涉TGF-β受体Ⅱ(TGF-βreceptorⅡ,TGFBR2)后,MTT检测TR乳腺癌细胞trastuzumab敏感性,transwell实验检测TR乳腺癌细胞侵袭力,q PCR检测EMT相关转录因子水平变化。通过mi RNA芯片分析筛选WT与TR乳腺癌细胞中显著差异表达的mi RNAs。MTT与流式细胞术凋亡检测分析mi R-200c对乳腺癌细胞trastuzumab敏感性的影响。Traswell与划痕实验分析mi R-200c对乳腺癌细胞侵袭与迁移能力的影响。形态学观察分析与WB检测分析mi R-200c对EMT形态及标记分子的影响。WB检测分析mi R-200c对乳腺癌细胞TGF-β信号的影响,q PCR与ELISA检测分析mi R-200c对TGF-β表达的影响。裸鼠体内成瘤与肺转移实验分析mi R-200c对乳腺癌在动物体内trastuzumab耐药与转移的影响。软件预测mi R-200c靶蛋白并使用双荧光素酶报告基因、q PCR及WB等实验进行靶标验证。MTT与流式细胞术凋亡检测验证靶蛋白对trastuzumab敏感性的影响,Traswell与划痕实验验证靶蛋白对乳腺癌细胞侵袭与迁移能力的影响,形态学观察靶蛋白对EMT形态影响,WB分析靶蛋白对EMT标记分子的影响。QPCR、WB与ELISA方法分析细胞中是否存在调控乳腺癌trastuzumab耐药的mi R-200c/ZEB1与mi R-200c/ZNF217/TGF-β/ZEB1巢式环路。【结果】通过对乳腺癌细胞施加5μg/m L trastuzumab持续药物筛选约6个月,成功建立体外TR乳腺癌细胞模型。相较于WT乳腺癌细胞,MTT结果表明TR乳腺癌细胞对trastuzumab敏感性显著减低;软琼脂克隆形成实验与transwell结果表明,TR乳腺癌细胞非锚定依赖生长能力与侵袭力显著增加;形态学观察、WB检测EMT标记物、q PCR检测EMT相关转录因子结果均表明TR乳腺癌细胞发生并维持明显EMT。相较于WT乳腺癌细胞,WB结果表明,TR乳腺癌细胞TGF-β信号激活水平显著升高;q PCR与ELISA结果表明,TR乳腺癌细胞TGF-βm RNA水平与分泌水平均显著升高。Si RNA干涉TGFBR2后,MTT结果表明TR乳腺癌细胞trastuzumab敏感性显著提高,transwell结果表明TR乳腺癌细胞侵袭力显著下降,q PCR结果表明TR乳腺癌细胞中EMT相关转录因子显著下调。Mi RNA芯片结果表明mi R-200c在TR乳腺癌细胞中降低最为显著。MTT与流式细胞术凋亡检测结果表明,mi R-200c可使TR乳腺癌细胞对trastuzumab敏感性显著提高;transwell与划痕实验结果表明,mi R-200c可显著降低TR乳腺癌细胞侵袭与迁移能力;形态学观察与WB结果表明,mi R-200c可有效逆转TR乳腺癌细胞EMT;WB结果表明,mi R-200c可显著抑制TGF-β信号;q PCR与ELISA结果表明,mi R-200c可显著下调TR乳腺癌细胞中TGF-β2与TGF-β3的m RNA水平与分泌蛋白水平。裸鼠体内成瘤与尾静脉肺转移实验结果表明,mi R-200c可在体内使TR乳腺癌细胞对trastuzumab敏感性提高并抑制其转移。软件预测、双荧光素酶报告基因检测、q PCR及WB结果表明,mi R-200c直接靶向锌指蛋白ZNF217与转录因子ZEB1。Si RNA干涉ZNF217与ZEB1均可逆转TR细胞trastuzumab耐药和EMT,并降低其侵袭与迁移能力。此外,q PCR、WB及ELISA结果表明乳腺癌中mi R-200c、ZNF217、TGF-β、ZEB1共同构成mi-R200c/ZEB1与mi R-200c/ZNF217/TGF-β/ZEB1巢式调节环路。【结论】本研究发现乳腺癌诱发trastuzumab耐药的同时,伴发EMT、非锚定依赖生长能力与侵袭能力的增强等恶性表型。TGF-β信号同时在乳腺癌trastuzumab耐药细胞的耐药性、EMT及侵袭力增强等多种恶性表型的诱导与维持中具有重要作用。通过进一步研究发现,mi R-200c通过靶向ZNF217与ZEB1调控TGF-β信号,逆转乳腺癌trastuzumab耐药并同时抑制乳腺癌转移。最后,我们还发现,在乳腺癌中,mi R-200c、ZNF217、TGF-β、ZEB1共同构成mi R-200c/ZEB1与mi R-200c/ZNF217/TGF-β/ZEB1巢式调节环路。这些研究结果阐释了调控乳腺癌恶性表型诱导与维持的新机制,并为其治疗提供了新的理论依据。
[Abstract]:[background] at present, breast cancer has become one of the most serious diseases threatening women's life major health, disease prevention and control is one of the hot issues in the field of cancer research. Human epidermal growth factor receptor 2 (human epidermalgrowth factor receptor-2, HER2) expression in 20% ~25% patients with breast cancer, the prognosis is poor, prone to metastasis HER2, has become an important target for treatment of breast cancer. Trastuzumab (trade name: Herceptin, trastuzumab, Hessaitin) is targeting a humanized HER2 monoclonal antibody, has achieved great success in the early treatment and treatment of metastatic HER2 positive breast cancer. However, the resistance of trastuzumab and the potential metastasis caused by increased it has been a problem for clinicians. In recent years, however, trastuzumab breast cancer drug resistance and metastasis of breast cancer research has made significant progress, breast cancer resistance and metastasis and trastuzumab The exact molecular mechanism still needs further elucidation of.TGF- beta signaling hand in tumor development plays an important role and played a dual role, its inhibition of proliferation and apoptosis in the early stage of tumor, on the other hand, it can effectively induce apoptosis of tumor cells and maintenance of epithelial mesenchymal transition in advanced cancer (epithelial-mesenchymal transition EMT), thereby promoting tumor invasion and metastasis. In addition, the role of TGF- beta signaling in tumor radiotherapy and drug resistance in the treatment of tumor gradually attracted attention, has become a hot research field. More and more evidence show that TGF- signaling plays an important role in tumor metastasis and drug resistance, is expected to become the important target for cancer therapy. However, about TGF- beta signaling in breast cancer with metastatic potential trastuzumab resistance and increase the effect and its regulation mechanism is not clear.Mi is RNAs A class of small molecule RNA protein participates in tumor growth, to regulation by the target development. Therefore, the potential effects and elucidate the molecular mechanism underlying Mi mediated TGF- beta RNA in signal regulation of HER2 positive breast cancer drug resistance and metastasis in trastuzumab, provides a new idea for the reversal of the malignant phenotype of breast cancer multiple, has important significance for the treatment of breast cancer. [Objective] to clarify the underlying mechanisms involved in the regulation of MI RNA and trastuzumab regulation of breast cancer resistance and transfer of TGF- signals, and provide a new way for the treatment of breast cancer metastasis or trastuzumab resistance. [method] the wild type culture through continuous dosing (wide type, WT) of breast cancer cells to establish in vitro breast cancer resistant to trastuzumab (trastuzumab resistant TR) cell model, MTT detection and analysis of breast cancer WT and TR breast cancer cells to trastuzumab drug sensitivity. Soft agar clone The formation of experimental analysis of anchorage independent growth ability of WT and TR in breast cancer cells; Transwell invasion experiment analysis of WT and TR in breast cancer cell invasion ability; to observe the morphology of WT and TR in breast cancer cells, quantitative real-time PCR (Quantitative realtime PCR, Q PCR) to detect EMT transcription factor levels, EMT molecular marker detection WB in order to observe the detection of breast cancer WT cells induced by EMT.WB and TR in breast cancer cells TGF- beta signaling activation, Q PCR and ELISA.RNA TGF- expression detection and analysis of beta interference TGF- beta receptor II (TGF- beta receptor II, TGFBR2), MTT detection of TR breast cancer cell invasion detection sensitivity of trastuzumab TR breast cancer cell line Transwell the change of Q PCR detection of EMT related transcription factor levels. Through the MI RNA chip analysis were significant difference between WT and TR expression in breast cancer cells mi RNAs.MTT and flow cytometry analysis of MI R-200c on apoptosis in breast cancer Analysis of the influence of MI R-200c on breast cancer cell invasion and migration effect of.Traswell and trastuzumab cell scratch assay sensitivity. Morphological analysis and WB detection and analysis of MI R-200c effect on the EMT morphology and molecular markers of.WB detection and analysis of the influence of MI R-200c on breast cancer cell TGF- beta signal, Q PCR and ELISA detection and analysis of effect of MI R-200c on the expression of TGF- beta. Nude mice experiment to analyze the effect of MI R-200c on trastuzumab in the animal drug resistance and metastasis of breast cancer tumor and lung metastasis. Mi R-200c software to predict the target protein and using dual luciferase reporter gene, Q PCR and WB.MTT experiment target validation effect and flow cytometry apoptosis detection test target protein sensitivity to trastuzumab, effects of Traswell and scratch test verification of target protein on breast cancer cell invasion and migration, morphological observation on the morphology of EMT target protein Influence of WB effect on the EMT of the target protein molecular markers.QPCR, WB and ELISA method to analyze whether there is regulation of breast cancer trastuzumab cells in MI R-200c/ZEB1 and MI R-200c/ZNF217/TGF- beta /ZEB1 nested loop. [results] the breast cancer cells 5 g/m L trastuzumab applied drug screening continued for about 6 months, the successful establishment of TR breast cancer cell model in vitro. Compared to WT breast cancer cells, MTT results show that the TR breast cancer cell sensitivity to trastuzumab decreased significantly; soft agar colony formation assay and Transwell results showed that the anchorage independent growth and invasiveness of TR breast cancer cells increased significantly; morphological observation, WB detection of EMT markers, Q PCR detection the results showed that EMT related transcription factor TR in breast cancer cells and maintain EMT. significantly compared to WT breast cancer cells, WB results showed that the TR breast cancer cell TGF- beta signaling activated water The flat was significantly increased; Q PCR and ELISA results showed that the TR TGF- breast cancer cell m beta RNA levels and secretion levels were significantly increased in.Si RNA interference TGFBR2, MTT results showed that TR trastuzumab significantly increased the sensitivity of breast cancer cells, Transwell results show that the TR breast cancer cell invasion ability decreased significantly, Q PCR results showed that TR breast cancer cells in the EMT related transcription factor significantly reduced.Mi RNA chip results showed that MI R-200c in TR breast cancer cells was reduced significantly and.MTT flow cytometry apoptosis detection results show that MI R-200c can make TR breast cancer cells sensitive to trastuzumab significantly increased; Transwell and scratch test results showed that MI R-200c can significantly decrease the TR of breast cancer cell invasion and migration ability; morphological observation and WB results showed that MI R-200c could effectively reverse the TR breast cancer cell EMT; WB results showed that MI R-200c could significantly inhibit the beta TGF- signal; Q PCR With the ELISA results shows that MI, R-200c could significantly decrease TR in breast cancer cells TGF- beta 2 and beta 3 TGF- m RNA level and protein level. The secretion of tumor formation in nude mice and the tail vein of lung metastasis. The experimental results show that MI R-200c can make TR breast cancer cells in vivo increased sensitivity to trastuzumab and inhibit the transfer of software. Prediction, dual luciferase reporter gene assay, Q PCR and WB Mi results showed that R-200c directly targeted zinc finger protein ZNF217 and transcription factor ZEB1.Si RNA interference ZNF217 and ZEB1 can reverse TR cell resistance to trastuzumab and EMT, and decrease the ability of invasion and migration. In addition, q, PCR, WB and ELISA results showed that MI in breast cancer R-200c, ZNF217, TGF-, ZEB1 beta, composed of the mi-R200c/ZEB1 and MI R-200c/ZNF217/TGF- beta /ZEB1 nested regulation loop. [Conclusion] this study found that trastuzumab induced resistance of breast cancer at the same time, with EMT, anchorage independent growth At the same time, drug resistance in trastuzumab resistant breast cancer cell invasion ability and enhancement of malignant phenotype of.TGF- beta signaling, EMT and invasiveness enhancement plays an important role in the induction and maintenance of a variety of malignant phenotype. Through further study found that MI R-200c by targeting ZNF217 and ZEB1 regulation of TGF- beta signaling, trastuzumab resistance and the reversal of breast cancer at the same time inhibit breast cancer metastasis. Finally, we also found that in breast cancer, MI R-200c, ZNF217 TGF-, ZEB1 beta, composed of the MI R-200c/ZEB1 and MI R-200c/ZNF217/TGF- beta /ZEB1 nested regulation loop. These research results explain the mechanism of the new regulation of malignant phenotype of breast cancer induction and maintenance, and provide a new theoretical basis for its treatment.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9
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本文编号：1745700