本文選題：miR-200c + TGF-β信號(hào)��； 參考：《第四軍醫(yī)大學(xué)》2015年博士論文

【摘要】：【背景】目前,乳腺癌已經(jīng)成為最為主要的威脅女性生命健康的惡性疾病之一,該病防治是腫瘤研究領(lǐng)域的熱點(diǎn)問題之一。人表皮生長因子受體2(human epidermalgrowth factor receptor-2,HER2)過表達(dá)于20%~25%乳腺癌,患者治療預(yù)后差,易發(fā)生轉(zhuǎn)移,HER2已經(jīng)成為治療乳腺癌的重要靶點(diǎn)。曲妥珠單抗(trastuzumab,商品名:herceptin,赫賽汀)是靶向HER2的人源化單克隆抗體,在HER2陽性乳腺癌早期治療及轉(zhuǎn)移治療中取得較大成功。然而,trastuzumab耐藥以及由此而導(dǎo)致的潛在轉(zhuǎn)移性增加卻一直困擾臨床醫(yī)生。近年來,乳腺癌trastuzumab耐藥與乳腺癌轉(zhuǎn)移研究取得顯著進(jìn)步,然而,乳腺癌trastuzumab耐藥與轉(zhuǎn)移相關(guān)性及其具體的分子機(jī)制尚需進(jìn)一步闡明。TGF-β信號(hào)在腫瘤發(fā)生發(fā)展中具有重要作用并扮演著雙重角色,一方面,其在腫瘤早期發(fā)揮增殖抑制與促凋亡作用,另一方面,其在腫瘤晚期可有效誘導(dǎo)腫瘤細(xì)胞發(fā)生并維持上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT),從而促進(jìn)腫瘤侵襲與轉(zhuǎn)移。此外,TGF-β信號(hào)在多種腫瘤放射與藥物治療抵抗中的作用逐漸引起重視,成為腫瘤研究領(lǐng)域的熱點(diǎn)。越來越多的證據(jù)表明,TGF-β信號(hào)在腫瘤耐藥與轉(zhuǎn)移中具有重要作用,有望成為重要的腫瘤治療靶點(diǎn)。然而,關(guān)于TGF-β信號(hào)在乳腺癌trastuzumab耐藥及伴隨潛在轉(zhuǎn)移性增加中的作用及其自身調(diào)控機(jī)制尚不明確。Mi RNAs是一類小分子RNA,通過靶向調(diào)控蛋白質(zhì)而廣泛參與腫瘤發(fā)生、發(fā)展。因此,闡明mi RNA在介導(dǎo)TGF-β信號(hào)調(diào)控HER2陽性乳腺癌trastuzumab耐藥和轉(zhuǎn)移中的潛在作用及其分子機(jī)制,將為乳腺癌多重惡性表型的逆轉(zhuǎn)提供新的思路,對(duì)于乳腺癌臨床治療具有重要意義�！灸康摹筷U明mi RNA參與TGF-β信號(hào)調(diào)節(jié)并調(diào)控乳腺癌trastuzumab耐藥與轉(zhuǎn)移的潛在作用機(jī)制,為trastuzumab耐藥或者轉(zhuǎn)移乳腺癌治療提供新的思路�！痉椒ā客ㄟ^持續(xù)加藥培養(yǎng)野生型(wide type,WT)乳腺癌細(xì)胞的方法建立體外乳腺癌trastuzumab耐藥(trastuzumab resistant,TR)細(xì)胞模型,MTT檢測分析乳腺癌WT與TR乳腺癌細(xì)胞對(duì)trastuzumab藥物敏感性。軟瓊脂克隆形成實(shí)驗(yàn)分析WT與TR乳腺癌細(xì)胞非錨定依賴生長能力;transwell侵襲實(shí)驗(yàn)分析WT與TR乳腺癌細(xì)胞侵襲能力;對(duì)WT與TR乳腺癌細(xì)胞進(jìn)行形態(tài)學(xué)觀察,實(shí)時(shí)定量PCR(quantitative realtime PCR,q PCR)檢測EMT轉(zhuǎn)錄因子水平,WB檢測EMT標(biāo)記分子,以觀察細(xì)胞誘發(fā)EMT。WB檢測乳腺癌WT與TR乳腺癌細(xì)胞TGF-β信號(hào)激活狀態(tài),q PCR與ELISA檢測分析TGF-β的表達(dá)。RNA干涉TGF-β受體Ⅱ(TGF-βreceptorⅡ,TGFBR2)后,MTT檢測TR乳腺癌細(xì)胞trastuzumab敏感性,transwell實(shí)驗(yàn)檢測TR乳腺癌細(xì)胞侵襲力,q PCR檢測EMT相關(guān)轉(zhuǎn)錄因子水平變化。通過mi RNA芯片分析篩選WT與TR乳腺癌細(xì)胞中顯著差異表達(dá)的mi RNAs。MTT與流式細(xì)胞術(shù)凋亡檢測分析mi R-200c對(duì)乳腺癌細(xì)胞trastuzumab敏感性的影響。Traswell與劃痕實(shí)驗(yàn)分析mi R-200c對(duì)乳腺癌細(xì)胞侵襲與遷移能力的影響。形態(tài)學(xué)觀察分析與WB檢測分析mi R-200c對(duì)EMT形態(tài)及標(biāo)記分子的影響。WB檢測分析mi R-200c對(duì)乳腺癌細(xì)胞TGF-β信號(hào)的影響,q PCR與ELISA檢測分析mi R-200c對(duì)TGF-β表達(dá)的影響。裸鼠體內(nèi)成瘤與肺轉(zhuǎn)移實(shí)驗(yàn)分析mi R-200c對(duì)乳腺癌在動(dòng)物體內(nèi)trastuzumab耐藥與轉(zhuǎn)移的影響。軟件預(yù)測mi R-200c靶蛋白并使用雙熒光素酶報(bào)告基因、q PCR及WB等實(shí)驗(yàn)進(jìn)行靶標(biāo)驗(yàn)證。MTT與流式細(xì)胞術(shù)凋亡檢測驗(yàn)證靶蛋白對(duì)trastuzumab敏感性的影響,Traswell與劃痕實(shí)驗(yàn)驗(yàn)證靶蛋白對(duì)乳腺癌細(xì)胞侵襲與遷移能力的影響,形態(tài)學(xué)觀察靶蛋白對(duì)EMT形態(tài)影響,WB分析靶蛋白對(duì)EMT標(biāo)記分子的影響。QPCR、WB與ELISA方法分析細(xì)胞中是否存在調(diào)控乳腺癌trastuzumab耐藥的mi R-200c/ZEB1與mi R-200c/ZNF217/TGF-β/ZEB1巢式環(huán)路�！窘Y(jié)果】通過對(duì)乳腺癌細(xì)胞施加5μg/m L trastuzumab持續(xù)藥物篩選約6個(gè)月,成功建立體外TR乳腺癌細(xì)胞模型。相較于WT乳腺癌細(xì)胞,MTT結(jié)果表明TR乳腺癌細(xì)胞對(duì)trastuzumab敏感性顯著減低;軟瓊脂克隆形成實(shí)驗(yàn)與transwell結(jié)果表明,TR乳腺癌細(xì)胞非錨定依賴生長能力與侵襲力顯著增加;形態(tài)學(xué)觀察、WB檢測EMT標(biāo)記物、q PCR檢測EMT相關(guān)轉(zhuǎn)錄因子結(jié)果均表明TR乳腺癌細(xì)胞發(fā)生并維持明顯EMT。相較于WT乳腺癌細(xì)胞,WB結(jié)果表明,TR乳腺癌細(xì)胞TGF-β信號(hào)激活水平顯著升高;q PCR與ELISA結(jié)果表明,TR乳腺癌細(xì)胞TGF-βm RNA水平與分泌水平均顯著升高。Si RNA干涉TGFBR2后,MTT結(jié)果表明TR乳腺癌細(xì)胞trastuzumab敏感性顯著提高,transwell結(jié)果表明TR乳腺癌細(xì)胞侵襲力顯著下降,q PCR結(jié)果表明TR乳腺癌細(xì)胞中EMT相關(guān)轉(zhuǎn)錄因子顯著下調(diào)。Mi RNA芯片結(jié)果表明mi R-200c在TR乳腺癌細(xì)胞中降低最為顯著。MTT與流式細(xì)胞術(shù)凋亡檢測結(jié)果表明,mi R-200c可使TR乳腺癌細(xì)胞對(duì)trastuzumab敏感性顯著提高;transwell與劃痕實(shí)驗(yàn)結(jié)果表明,mi R-200c可顯著降低TR乳腺癌細(xì)胞侵襲與遷移能力;形態(tài)學(xué)觀察與WB結(jié)果表明,mi R-200c可有效逆轉(zhuǎn)TR乳腺癌細(xì)胞EMT;WB結(jié)果表明,mi R-200c可顯著抑制TGF-β信號(hào);q PCR與ELISA結(jié)果表明,mi R-200c可顯著下調(diào)TR乳腺癌細(xì)胞中TGF-β2與TGF-β3的m RNA水平與分泌蛋白水平。裸鼠體內(nèi)成瘤與尾靜脈肺轉(zhuǎn)移實(shí)驗(yàn)結(jié)果表明,mi R-200c可在體內(nèi)使TR乳腺癌細(xì)胞對(duì)trastuzumab敏感性提高并抑制其轉(zhuǎn)移。軟件預(yù)測、雙熒光素酶報(bào)告基因檢測、q PCR及WB結(jié)果表明,mi R-200c直接靶向鋅指蛋白ZNF217與轉(zhuǎn)錄因子ZEB1。Si RNA干涉ZNF217與ZEB1均可逆轉(zhuǎn)TR細(xì)胞trastuzumab耐藥和EMT,并降低其侵襲與遷移能力。此外,q PCR、WB及ELISA結(jié)果表明乳腺癌中mi R-200c、ZNF217、TGF-β、ZEB1共同構(gòu)成mi-R200c/ZEB1與mi R-200c/ZNF217/TGF-β/ZEB1巢式調(diào)節(jié)環(huán)路�！窘Y(jié)論】本研究發(fā)現(xiàn)乳腺癌誘發(fā)trastuzumab耐藥的同時(shí),伴發(fā)EMT、非錨定依賴生長能力與侵襲能力的增強(qiáng)等惡性表型。TGF-β信號(hào)同時(shí)在乳腺癌trastuzumab耐藥細(xì)胞的耐藥性、EMT及侵襲力增強(qiáng)等多種惡性表型的誘導(dǎo)與維持中具有重要作用。通過進(jìn)一步研究發(fā)現(xiàn),mi R-200c通過靶向ZNF217與ZEB1調(diào)控TGF-β信號(hào),逆轉(zhuǎn)乳腺癌trastuzumab耐藥并同時(shí)抑制乳腺癌轉(zhuǎn)移。最后,我們還發(fā)現(xiàn),在乳腺癌中,mi R-200c、ZNF217、TGF-β、ZEB1共同構(gòu)成mi R-200c/ZEB1與mi R-200c/ZNF217/TGF-β/ZEB1巢式調(diào)節(jié)環(huán)路。這些研究結(jié)果闡釋了調(diào)控乳腺癌惡性表型誘導(dǎo)與維持的新機(jī)制,并為其治療提供了新的理論依據(jù)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9
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本文編號(hào)：1745700