本文選題：乳腺腫瘤 + HOTAIR　； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：目的：研究及初步探討HOTAIR與乳腺癌曲妥珠單抗耐藥的關(guān)系及其可能的分子機(jī)制。方法：(1)選取乳腺癌細(xì)胞系SK-BR-3-TS,通過間歇大劑量沖擊和逐步增加劑量相結(jié)合的方法,誘導(dǎo)建立曲妥珠單抗耐藥細(xì)胞系SK-BR-3-TR。(2) qRT-PCR檢測(cè)SK-BR-3-TS和SK-BR-3-TR細(xì)胞HOTAIR的表達(dá),MTT法檢測(cè)兩種細(xì)胞模型腫瘤細(xì)胞的增殖活性,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡,transwell侵襲實(shí)驗(yàn)觀察細(xì)胞侵襲能力,qRT-PCR和western blot分別檢測(cè)細(xì)胞上皮間質(zhì)轉(zhuǎn)化(epithelial mesenchymal transition, EMT)相關(guān)基因和蛋白TGF-β、Snail、 Vimentin和E-cadherin的表達(dá)以及PI3K/AKT/mTOR和MEK/MAPK信號(hào)通路活性。(3)甲基化特異性PCR (Methylation-Specific PCR, MSP)檢測(cè)兩種細(xì)胞TGF-β、 PTEN及CNK1B(P27kip1)基因甲基化水平。(4)慢病毒轉(zhuǎn)染siRNA干擾實(shí)驗(yàn)阻抑SK-BR-3-TR細(xì)胞HOTAIR表達(dá),反向驗(yàn)證HOTAIR與曲妥珠單抗耐藥的關(guān)系及其分子機(jī)制。(5) BALB/c裸鼠荷瘤體內(nèi)實(shí)驗(yàn),分別建立SK-BR-3-TS、SK-BR-3-TR和si-HOTAIR-SK-BR-3-TR裸鼠荷瘤模型,觀察三種模型瘤體生長(zhǎng)速度；免疫組化技術(shù)檢測(cè)瘤體TGF-β、Snail和E-cadherin以及PTEN、Ki67的表達(dá)水平。體內(nèi)實(shí)驗(yàn)進(jìn)一步驗(yàn)證HOTAIR對(duì)曲妥珠單抗耐藥的影響。結(jié)果：(1)在成功誘導(dǎo)及穩(wěn)定培養(yǎng)乳腺癌曲妥珠單抗耐藥細(xì)胞系SK-BR-3-TR中,與曲妥珠單抗敏感細(xì)胞株SK-BR-3-TS相比,HOT AIR RNA表達(dá)水平明顯上調(diào)(2.216±0.332 v 0.326±0.05,P=0.0006)。(2)MTT法、transwell侵襲實(shí)驗(yàn)及流式細(xì)胞術(shù)檢測(cè)結(jié)果提示,與敏感細(xì)胞相比,耐藥細(xì)胞增殖活性及侵襲能力增強(qiáng),細(xì)胞凋亡減少。(3) qRT-PCR及WB檢測(cè)顯示,與敏感細(xì)胞相比,耐藥細(xì)胞EMT相關(guān)基因TGF-β、 Snail及Vimentin表達(dá)上調(diào),E-cadherin表達(dá)下調(diào),HER2受體信號(hào)通路中HER2、 PI3K、AK、mTOR及MAPK基因表達(dá)水平及HER2受體磷酸化水平無明顯變化,但p-AKT、p-MAPK及CyclinD1水平升高,而PTEN、P27水平下調(diào)。(4)MSP檢測(cè)耐藥細(xì)胞PTEN基因甲基化水平升高,TGF-β基因甲基化水平下調(diào),而CNK1B(P27kip1)基因甲基化水平未發(fā)生明顯變化。(5)慢病毒介導(dǎo)RNA干擾實(shí)驗(yàn)阻抑耐藥細(xì)胞HOTAIR表達(dá)后,HOTAIR表達(dá)明顯下調(diào)。MTT法檢測(cè)siHOTAIR-SK-BR-3-TR細(xì)胞恢復(fù)了對(duì)曲妥珠單抗的敏感性。細(xì)胞增殖活性及侵襲能力減弱,細(xì)胞凋亡比例增加,qRT-PCR及WB檢測(cè)細(xì)胞EMT相關(guān)基因及蛋白TGF-β、Snail及Vimentin表達(dá)下調(diào),E-cadherin表達(dá)上調(diào),HER2受體信號(hào)通路中HER2、PI3K、AKT、mTOR及MAPK表達(dá)水平及HER2磷酸化水平未發(fā)生明顯改變,但p-AKT、p-MAPK及CyclinD1水平下調(diào),而PTEN、P27水平上調(diào)。(6)MSP檢測(cè)siHOTAIR-SK-BR-3-TR細(xì)胞PTEN基因甲基化水平下調(diào),TGF-β基因甲基化水平上調(diào),而CNK1B(P27kip1)基因甲基化水平無顯著差異。(7)成功建立SK-BR-3-TS、SK-BR-3-TR和si-HOTAIR-SK-BR-3-TR裸鼠荷瘤模型。SK-BR-3-TR荷瘤模型瘤體生長(zhǎng)速度較SK-BR-3-TS和si-HOTAIR-SK-BR-3-TR模型明顯增快,差異具有統(tǒng)計(jì)學(xué)意義(P.05)。si-HOTAIR-SK-BR-3-TR模型瘤體TGF-β和Snail蛋白陽性表達(dá)率(1/6,16.7%；2/6,33.3%)低于SK-BR-3-TR(4/6,66.7%; 5/6,83.3%),但差異無統(tǒng)計(jì)學(xué)意義(Chi-Square tests,Fisher's Exact Test, P=0.242); E-cadherin及PTEN蛋白陽性表達(dá)率較高,但無統(tǒng)計(jì)學(xué)差異(P=0.242)。Ki67的表達(dá)陽性率無差別(均為5/6,83.3%,p=1.000)。結(jié)論：曲妥珠單抗耐藥細(xì)胞株SK-BR-3-TR中HOTAIR表達(dá)上調(diào)誘導(dǎo)的TGF-β去甲基化表觀遺傳修飾及PTEN基因甲基化效應(yīng),從而分別導(dǎo)致耐藥細(xì)胞EMT及PI3K/AKT/mTOR通路內(nèi)在活性增強(qiáng)。HAOTAIR的雙重表觀遺傳調(diào)控機(jī)制可能參與了SK-BR-3-TR細(xì)胞對(duì)曲妥珠單抗耐藥。靶向抑制HOTAIR表達(dá)可能逆轉(zhuǎn)曲妥珠單抗耐藥。
[Abstract]:Objective: To study and preliminarily explore the relationship between HOTAIR and the resistance of trastuzumab in breast cancer and its possible molecular mechanism. Methods: (1) select the breast cancer cell line SK-BR-3-TS, through the intermittent large dose impact and gradually increase the dose combination method, to induce the establishment of SK-BR-3-TR. (2) qRT-PCR of trastuzumab resistant cell line to detect SK-BR-3-T The expression of HOTAIR in S and SK-BR-3-TR cells, MTT method was used to detect the proliferation activity of two cell model tumor cells. Flow cytometry was used to detect cell apoptosis. Transwell invasion test was used to observe cell invasiveness. QRT-PCR and Western blot were used to detect epithelial mesenchymal transition (epithelial mesenchymal transition, EMT) related genes and protein beta The expression of Snail, Vimentin and E-cadherin as well as the activity of PI3K/AKT/mTOR and MEK/MAPK signaling pathway. (3) methylation specific PCR (Methylation-Specific PCR, MSP) detection of the methylation level of two cells TGF- beta, PTEN and CNK1B genes. (4) lentivirus transfection interference experiment blocking expression, reverse validation The relationship between R and resistance to trastuzumab and its molecular mechanism. (5) BALB/c nude mice were loaded with tumor model in nude mice, and the growth rate of three models of tumor body was observed in SK-BR-3-TS, SK-BR-3-TR and si-HOTAIR-SK-BR-3-TR nude mice. Immunohistochemistry technique was used to detect the expression level of TGF- beta, Snail and E-cadherin, PTEN, Ki67. The effect of HOTAIR on the resistance of trastuzumab was further verified. Results: (1) the expression level of HOT AIR RNA was significantly up-regulated (2.216 + 0.332 V 0.326 + 0.05, P=0.0006) in the successful induction and stabilization of breast cancer trastuzumab resistant cell line SK-BR-3-TR, compared with SK-BR-3-TS of trastuzumab sensitive cell strain (P=0.0006). (2) MTT method, Transwell invasion The results of the test and flow cytometry showed that the proliferation and invasion ability of drug-resistant cells were enhanced and apoptosis decreased compared with sensitive cells. (3) qRT-PCR and WB detection showed that the EMT related gene TGF- beta, Snail and Vimentin up regulation, E-cadherin expression down and HER in HER2 receptor signaling pathway were compared with sensitive cells. 2, PI3K, AK, mTOR and MAPK gene expression level and HER2 receptor phosphorylation level did not change significantly, but p-AKT, p-MAPK and CyclinD1 levels increased, and PTEN, P27 levels down. (4) MSP detection of drug resistance cell PTEN genes increased the level of methylated under the level of methylated gene, but the level of methylation of the gene was not significantly changed. (5 Lentivirus mediated RNA interference test inhibited the expression of HOTAIR in drug-resistant cells, the expression of HOTAIR was obviously down regulated by.MTT method and the sensitivity of siHOTAIR-SK-BR-3-TR cells to trastuzumab was restored. The cell proliferation activity and invasion ability were weakened, the proportion of cell apoptosis was increased. QRT-PCR and WB were used to detect EMT related genes and protein TGF- beta, Snail and Vime. The expression of ntin was down, and the expression of E-cadherin was up-regulated. The expression level of HER2, PI3K, AKT, mTOR and MAPK in the HER2 receptor signaling pathway and the level of HER2 phosphorylation were not significantly changed, but p-AKT, p-MAPK and CyclinD1 levels were down regulated. (6) the methylation of the gene was detected. There was no significant difference in the level of CNK1B (P27kip1) gene methylation. (7) successful establishment of SK-BR-3-TS, SK-BR-3-TR and si-HOTAIR-SK-BR-3-TR nude mice bearing tumor model.SK-BR-3-TR tumor model tumor growth speed was significantly faster than the SK-BR-3-TS and si-HOTAIR-SK-BR-3-TR model, the difference was statistically significant (P.05).Si-HOTAIR-SK-BR-3-TR model. The positive rate of TGF- beta and Snail protein (1/6,16.7%; 2/6,33.3%) was lower than SK-BR-3-TR (4/6,66.7%; 5/6,83.3%), but the difference was not statistically significant (Chi-Square tests, Fisher's Exact Test), but there was no difference in the positive rate of the protein (Chi-Square tests, Fisher's Exact Test). 83.3%, p=1.000) conclusion: TGF- beta demethylation epigenetic modification and PTEN gene methylation effect induced by the up regulation of HOTAIR expression in the resistant cell line SK-BR-3-TR of trastuzumab resistant cell line, which may lead to the double epigenetic regulation mechanism of the intrinsic activity enhancement.HAOTAIR in the EMT and PI3K/AKT/mTOR pathway of drug resistant cells, may be involved in SK-BR-3-TR Cells resistant to trastuzumab. Targeted inhibition of HOTAIR expression may reverse the resistance of trastuzumab.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9

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2 李望;HER2陽性復(fù)發(fā)轉(zhuǎn)移性乳腺癌曲妥珠單抗聯(lián)合長(zhǎng)春瑞濱或卡培他濱的療效及與p53基因相關(guān)性研究[D];安徽醫(yī)科大學(xué);2015年

3 曹敏;曲妥珠單抗、拉帕替尼及二者聯(lián)合用于乳腺癌新輔助治療的Meta分析[D];新疆醫(yī)科大學(xué);2015年

4 苗鳳真;曲妥珠單抗—納米金探針制備及對(duì)乳腺癌細(xì)胞體外生長(zhǎng)抑制作用[D];東南大學(xué);2015年

5 邵婉婷;曲妥珠單抗在Her-2陽性乳腺癌患者中的療效評(píng)價(jià)及相關(guān)預(yù)后影響因素分析[D];吉林大學(xué);2016年

6 吳夢(mèng)莞;PI3K/AKT通路的激活促進(jìn)HER2陽性胃癌曲妥珠單抗的耐藥[D];南方醫(yī)科大學(xué);2016年

7 阮貝鴻;探索乳腺癌細(xì)胞系中曲妥珠單抗耐藥相關(guān)的生物標(biāo)志物[D];浙江大學(xué);2016年

8 付明娜;曲妥珠單抗對(duì)乳腺癌術(shù)后輔助治療患者早期心臟毒性的觀察[D];青島大學(xué);2016年

9 關(guān)碩;曲妥珠單抗一線治療Her-2陽性轉(zhuǎn)移性乳腺癌的臨床觀察[D];大連醫(yī)科大學(xué);2016年

10 李慧;曲妥珠單抗聯(lián)合西妥昔單抗在耐藥胃癌中的抗腫瘤作用及其機(jī)理[D];中國(guó)人民解放軍醫(yī)學(xué)院;2014年

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