本文選題：乳腺癌　切入點(diǎn)：miR-181a　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景： 在我國,乳腺癌已經(jīng)成為女性發(fā)病率最高的惡性腫瘤。大約2/3的乳腺癌是雌激素受體(ER)和/或孕激素受體(PR)陽性,其生長具有雌激素依賴性�？勾萍に刂委熓且豁�(xiàng)有效、低毒的治療,其中特異性雌激素受體調(diào)節(jié)劑他莫昔芬tamoxifen(TAM),它阻滯了雌激素與受體的結(jié)合,目前已經(jīng)是內(nèi)分泌治療的金標(biāo)準(zhǔn)。大規(guī)模的臨床隨機(jī)試驗(yàn)表明,在早期ER陽性的乳腺癌患者中,術(shù)后5年tamoxifen的治療,減少了51%的復(fù)發(fā)和28%的死亡。但是,并非所有ER陽性和/或PR陽性的乳腺癌的病人都對(duì)tamoxifen治療有效,約40%的ER陽性乳腺癌病人一開始就對(duì)tamoxifen治療不敏感,另外,部分患者初始治療有效,長期使用后出現(xiàn)耐藥[5]。到目前為止,內(nèi)分泌療耐藥的確切機(jī)制尚不明確。微小RNA (MicroRNA,miRNA)是近年來發(fā)現(xiàn)的長度約為22nt的內(nèi)源性短鏈RNA,不編碼蛋白質(zhì),可通過與編碼蛋白質(zhì)的mRNA互補(bǔ)結(jié)合,沉默其表達(dá),從而起著調(diào)控細(xì)胞分化、生長、增殖、代謝、凋亡等功能。研究發(fā)現(xiàn)多種微小RNA與乳腺癌的發(fā)生、發(fā)展、治療耐藥等相關(guān)；還有部分微小RNA與E2和ER的信號(hào)傳導(dǎo)通路相關(guān)。 我們對(duì)209例的乳腺癌組織進(jìn)行microRNA基因芯片檢測分析,發(fā)現(xiàn)其中5個(gè)microRNA與ER陽性乳腺癌的生存相關(guān)。然后用RT-PCR方法檢測上述5個(gè)microRNA在他莫昔芬耐藥乳腺癌病人和非耐藥病人中的表達(dá)差異,發(fā)現(xiàn)miR-181a在耐藥病人中升高。因此,我們推測miR-181a可能與乳腺癌的內(nèi)分泌耐藥相關(guān)。我們擬通過體外研究探索miR-181a在他莫昔芬耐藥乳腺癌細(xì)胞中的表達(dá)情況,及其對(duì)內(nèi)分泌耐藥的影響,探討其可能的分子機(jī)制,驗(yàn)證miR-181a與ER陽性乳腺癌與預(yù)后的關(guān)系,以期找到內(nèi)分泌治療耐藥的預(yù)測方法和潛在治療靶點(diǎn) 方法和結(jié)果： 1. miR-181a在乳腺癌細(xì)胞株及他莫昔芬耐藥細(xì)胞株中的表達(dá)和對(duì)耐藥的影響 我們定量RT-PCR方法檢測了SK-BR-3、MDA231、T47D、MCF-7/W、 MCF-7/R-TAM、Bcap-37等乳腺癌細(xì)胞株中的miR-181a的表達(dá)水平,發(fā)現(xiàn)ER陰性的乳腺癌細(xì)胞中的miR-181a明顯升高。在ER陰性、Her-2陽性的SK-BR-3細(xì)胞的miR-181a的表達(dá)較其它ER陰性的乳腺癌細(xì)胞更高。我們用他莫昔芬敏感的MCF-7細(xì)胞株(MCF-7/W)建立一株他莫昔芬耐藥的乳腺癌細(xì)胞株(MCF-7/R-TAM),分別檢測兩株細(xì)胞miR-181a的表達(dá)水平,結(jié)果提示MCF-7/R-TAM細(xì)胞中miR-181a的表達(dá)明顯升高。將pre-miR-181a轉(zhuǎn)染細(xì)胞,結(jié)果顯示轉(zhuǎn)染pre-miR-181a后MCF-7/W細(xì)胞株的miR-181a明顯升高,相應(yīng)對(duì)他莫昔芬的敏感性降低。將anti-miR-181a轉(zhuǎn)染MCF-7/R-TAM細(xì)胞,其miR-181a降低,同時(shí)對(duì)他莫昔芬的敏感性增加。 2. miR-181a影響內(nèi)分泌治療耐藥的相關(guān)機(jī)制研究 為明確miR-181a與ER是否存在調(diào)控關(guān)系,我們用ER的消除劑氟維司群降低ER陽性的乳腺癌細(xì)胞株中的ER表達(dá),miR-181a的表達(dá)量隨之上升；將ER基因?qū)隕R陰性乳腺癌細(xì)胞Bcap-37形成穩(wěn)定表達(dá)ER的Bcap-37(ER陽性)細(xì)胞株,發(fā)現(xiàn)Bcap-37(ER陽性)的miR-181a表達(dá)則相應(yīng)下降。 分別調(diào)節(jié)MCF-7/W和MCF-7/R-TAM細(xì)胞的miR-181a表達(dá)后,檢測ER、Her-2、 mTOR和PI3K的表達(dá)。結(jié)果顯示調(diào)高miR-181a表達(dá)后mTOR的表達(dá)增強(qiáng),降低miR-181a表達(dá)后mTOR的表達(dá)減弱,而ER、Her-2和P13K表達(dá)無明顯改變。研究結(jié)果表明miR-181a不能調(diào)控ER表達(dá),其對(duì)乳腺癌他莫昔芬耐藥的影響可能是通過調(diào)節(jié)mTOR起作用的。 3. miR-181a表達(dá)水平與ER陽性乳腺癌預(yù)后的相關(guān)性分析 選取1998年至2005年我科收治的早期浸潤性乳腺癌患者共80例。提取乳腺癌石蠟標(biāo)本中的總RNA,然后用qRT-PCR檢測]miR-181a的表達(dá)量,最終有76例病人的數(shù)據(jù)用于分析。生存分析結(jié)果顯示miR-181a與乳腺癌的預(yù)后相關(guān),miR-181a表達(dá)較高的乳腺癌預(yù)后較差。進(jìn)一步分析顯示ER陽性乳腺癌中miR-181a對(duì)生存有很好的預(yù)測作用,具有統(tǒng)計(jì)學(xué)意義。多因素COX回歸分析表明miR-181a、內(nèi)分泌治療方式、腫瘤大小和淋巴結(jié)轉(zhuǎn)移與乳腺癌預(yù)后相關(guān),具有統(tǒng)計(jì)學(xué)意義。提示miR-181a是乳腺癌預(yù)后獨(dú)立預(yù)測因子。雙變量相關(guān)性分析顯示miR-181a表達(dá)與年齡、ER、月經(jīng)狀態(tài)呈負(fù)相關(guān)性,而與其他因素?zé)o明顯相關(guān)性,并且統(tǒng)計(jì)學(xué)有顯著意義。 結(jié)論： 體外實(shí)驗(yàn)表明miR-181a在他莫昔芬耐藥的乳腺癌細(xì)胞中表達(dá)明顯升高。降低乳腺癌細(xì)胞的miR-181a表達(dá)后,對(duì)他莫昔芬的敏感性明顯升高。進(jìn)一步研究顯示miR-181a表達(dá)受ER表達(dá)的影響；miR-181a可以促進(jìn)mTOR的表達(dá),但對(duì)ER, Her-2和PI3K表達(dá)無明顯影響。臨床乳腺癌標(biāo)本檢測發(fā)現(xiàn)miR181a的表達(dá)水平與乳腺癌患者的預(yù)后呈負(fù)相關(guān),是ER陽性的乳腺癌預(yù)后獨(dú)立的負(fù)性預(yù)測因子；本研究數(shù)據(jù)中miR-181a的表達(dá)水平與年齡、ER、月經(jīng)狀態(tài)呈負(fù)相關(guān)性。因此,miR-181a可以作為預(yù)測ER陽性乳腺癌預(yù)后的潛在指標(biāo)。
[Abstract]:Background:
In China, breast cancer has become the highest incidence of female malignant tumors. About 2/3 of breast cancer that is estrogen receptor (ER) and / or progesterone receptor (PR) positive, the growth of estrogen dependent. Anti estrogen therapy is an effective treatment, low toxicity, including specific estrogen receptor modulators tamoxifen tamoxifen (TAM), which block the estrogen receptor and is currently the gold standard endocrine treatment. That large-scale randomized clinical trials, in early-stage ER positive breast cancer, treatment tamoxifen 5 years after operation, 51% reduction in recurrence and 28% died. However, not all ER positive and / or PR positive breast cancer patients are effective in treatment of tamoxifen, ER positive breast cancer patients about 40% of the beginning is not sensitive to tamoxifen treatment, in addition, the effective part of patients with the initial treatment and drug resistance of [5 appeared after long-term use "So far, the exact mechanism of endocrine therapy resistance is not clear. The small RNA (MicroRNA, miRNA) is a newly discovered endogenous short chain length is about RNA 22nt, not by encoding proteins, and encoding protein mRNA complementary with its silencing, which plays a regulation of cell differentiation, growth. The proliferation, metabolism, apoptosis and other functions. The study found many small RNA and breast cancer occurrence, development, treatment and other related drug resistance; and the signal transduction pathway of RNA and E2 and tiny ER.
We studied 209 cases of breast cancer microRNA gene microarray analysis, found that 5 of microRNA and ER positive breast cancer survival. Then use the RT-PCR method to detect the expression of microRNA 5 in tamoxifen resistant breast cancer patients and non resistant patients, found that miR-181a increased in resistant patients. Therefore, endocrine the resistance we speculate that miR-181a may be associated with breast cancer. We conducted in vitro to explore the expression of miR-181a in tamoxifen resistant breast cancer cells, and its influence on endocrine resistance, to explore its possible molecular mechanism, the relationship between miR-181a and ER positive breast cancer prognosis and verification, in order to find the endocrine therapy resistance prediction method and potential therapeutic targets
Methods and results:
Expression of 1. miR-181a in breast cancer cell lines and tamoxifen resistant cell lines and its effect on drug resistance
Our quantitative RT-PCR method to detect the SK-BR-3, MDA231, T47D, MCF-7/W, MCF-7/R-TAM, Bcap-37 expression in breast cancer cell lines miR-181a, ER negative breast cancer cells in miR-181a was increased. In ER negative, Her-2 positive expression of SK-BR-3 cells of miR-181a than other ER negative breast cancer cells high. We used MCF-7 cells sensitive to tamoxifen (MCF-7/W) breast cancer cell lines to establish a tamoxifen resistant (MCF-7/R-TAM), the expression level of miR-181a in the two cell lines were detected. The results suggest that the expression of miR-181a and MCF-7/R-TAM cells increased significantly. The results showed that pre-miR-181a transfected cells, MCF-7/W cells transfected with miR-181a was significantly increased after pre-miR-181a, the corresponding sensitivity to tamoxifen decreased. Anti-miR-181a were transfected into MCF-7/R-TAM cells, the miR-181a decreased, while the tamoxifen sensitive to him Sex increases.
Study on the mechanism of 2. miR-181a affecting drug resistance in endocrine therapy
To determine the miR-181a and ER are regulatory relationships, we use ER to eliminate agent fulvestrant reduce ER positive breast cancer cell lines of ER expression, the expression of miR-181a was increased; the introduction of ER gene into ER negative breast cancer cell Bcap-37 to form a stable expression of ER Bcap-37 (ER positive) cells were found Bcap-37 (ER positive) miR-181a expression decreased correspondingly.
The expression of MCF-7/W and MCF-7/R-TAM cells were adjusted after miR-181a, detection of ER, Her-2, expression of mTOR and PI3K. The results showed that high expression of miR-181a enhanced the expression of mTOR, reduce the expression of miR-181a mTOR and ER, Her-2 expression decreased, and P13K showed no significant change. The results show that miR-181a can regulate the expression of ER, the the impact of breast cancer tamoxifen resistance may be through the regulation of mTOR function.
Correlation analysis of 3. miR-181a expression level and prognosis of ER positive breast cancer
From 1998 to 2005 in our department were early invasive breast cancer patients were 80 cases. Total RNA was extracted from paraffin embedded specimens of breast cancer, and the expression of qRT-PCR was detected by]miR-181a, the final 76 patients had data for analysis. Survival analysis showed that the prognosis of miR-181a is associated with breast cancer, the expression of miR-181a in breast cancer prognosis poor high. Further analysis showed that miR-181a ER positive breast cancer is a good predictor of survival, with statistical significance. COX regression analysis showed that miR-181a, endocrine therapy, the prognosis of tumor size and lymph node metastasis associated with breast cancer, with statistical significance. It is suggested that miR-181a is independent predictor of prognosis of breast cancer. Bivariate correlation analysis showed that the expression of miR-181a and ER, there was a negative correlation between age, menstrual status, and no significant correlation with other factors, and statistically significant Significance.
Conclusion:
In vitro experiments showed that the expression of miR-181a in breast cancer cells tamoxifen resistance increased significantly. The decreased expression of miR-181a breast cancer cells, sensitivity to tamoxifen increased significantly. Further studies showed that the expression of miR-181a affected the expression of ER; miR-181a can promote the expression of mTOR, but for ER, Her-2 and PI3K expression had no significant effect on the detection of clinical. Breast cancer samples were found to be negatively correlated with the expression level of miR181a and the prognosis of patients with breast cancer, ER negative breast cancer prognosis positive independent predictors; this study expression and age data in miR-181a ER, there was a negative correlation between the menstrual state. Therefore, miR-181a can be used as a potential prognostic index for predicting ER positive breast cancer.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 周玨宇;馬文麗;費(fèi)嘉;丁大鵬;石嶸;姜立;鄭文嶺;;微RNA對(duì)K562細(xì)胞基因表達(dá)譜的影響[J];南方醫(yī)科大學(xué)學(xué)報(bào);2006年05期

2 徐迎春;林玉梅;張鳳春;;乳腺癌內(nèi)分泌治療耐藥機(jī)制研究進(jìn)展[J];中華腫瘤防治雜志;2006年03期

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本文編號(hào)：1644089