miR-181a在ER阳性乳腺癌内分泌治疗耐药中的作用及对预后的预测价值

发布时间：2018-03-21 13:39

本文选题：乳腺癌　切入点：miR-181a　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：背景：在我国,乳腺癌已经成为女性发病率最高的恶性肿瘤。大约2/3的乳腺癌是雌激素受体(ER)和/或孕激素受体(PR)阳性,其生长具有雌激素依赖性。抗雌激素治疗是一项有效、低毒的治疗,其中特异性雌激素受体调节剂他莫昔芬tamoxifen(TAM),它阻滞了雌激素与受体的结合,目前已经是内分泌治疗的金标准。大规模的临床随机试验表明,在早期ER阳性的乳腺癌患者中,术后5年tamoxifen的治疗,减少了51%的复发和28%的死亡。但是,并非所有ER阳性和/或PR阳性的乳腺癌的病人都对tamoxifen治疗有效,约40%的ER阳性乳腺癌病人一开始就对tamoxifen治疗不敏感,另外,部分患者初始治疗有效,长期使用后出现耐药[5]。到目前为止,内分泌疗耐药的确切机制尚不明确。微小RNA (MicroRNA,miRNA)是近年来发现的长度约为22nt的内源性短链RNA,不编码蛋白质,可通过与编码蛋白质的mRNA互补结合,沉默其表达,从而起着调控细胞分化、生长、增殖、代谢、凋亡等功能。研究发现多种微小RNA与乳腺癌的发生、发展、治疗耐药等相关；还有部分微小RNA与E2和ER的信号传导通路相关。我们对209例的乳腺癌组织进行microRNA基因芯片检测分析,发现其中5个microRNA与ER阳性乳腺癌的生存相关。然后用RT-PCR方法检测上述5个microRNA在他莫昔芬耐药乳腺癌病人和非耐药病人中的表达差异,发现miR-181a在耐药病人中升高。因此,我们推测miR-181a可能与乳腺癌的内分泌耐药相关。我们拟通过体外研究探索miR-181a在他莫昔芬耐药乳腺癌细胞中的表达情况,及其对内分泌耐药的影响,探讨其可能的分子机制,验证miR-181a与ER阳性乳腺癌与预后的关系,以期找到内分泌治疗耐药的预测方法和潜在治疗靶点方法和结果： 1. miR-181a在乳腺癌细胞株及他莫昔芬耐药细胞株中的表达和对耐药的影响我们定量RT-PCR方法检测了SK-BR-3、MDA231、T47D、MCF-7/W、 MCF-7/R-TAM、Bcap-37等乳腺癌细胞株中的miR-181a的表达水平,发现ER阴性的乳腺癌细胞中的miR-181a明显升高。在ER阴性、Her-2阳性的SK-BR-3细胞的miR-181a的表达较其它ER阴性的乳腺癌细胞更高。我们用他莫昔芬敏感的MCF-7细胞株(MCF-7/W)建立一株他莫昔芬耐药的乳腺癌细胞株(MCF-7/R-TAM),分别检测两株细胞miR-181a的表达水平,结果提示MCF-7/R-TAM细胞中miR-181a的表达明显升高。将pre-miR-181a转染细胞,结果显示转染pre-miR-181a后MCF-7/W细胞株的miR-181a明显升高,相应对他莫昔芬的敏感性降低。将anti-miR-181a转染MCF-7/R-TAM细胞,其miR-181a降低,同时对他莫昔芬的敏感性增加。 2. miR-181a影响内分泌治疗耐药的相关机制研究为明确miR-181a与ER是否存在调控关系,我们用ER的消除剂氟维司群降低ER阳性的乳腺癌细胞株中的ER表达,miR-181a的表达量随之上升；将ER基因导入ER阴性乳腺癌细胞Bcap-37形成稳定表达ER的Bcap-37(ER阳性)细胞株,发现Bcap-37(ER阳性)的miR-181a表达则相应下降。分别调节MCF-7/W和MCF-7/R-TAM细胞的miR-181a表达后,检测ER、Her-2、 mTOR和PI3K的表达。结果显示调高miR-181a表达后mTOR的表达增强,降低miR-181a表达后mTOR的表达减弱,而ER、Her-2和P13K表达无明显改变。研究结果表明miR-181a不能调控ER表达,其对乳腺癌他莫昔芬耐药的影响可能是通过调节mTOR起作用的。 3. miR-181a表达水平与ER阳性乳腺癌预后的相关性分析选取1998年至2005年我科收治的早期浸润性乳腺癌患者共80例。提取乳腺癌石蜡标本中的总RNA,然后用qRT-PCR检测]miR-181a的表达量,最终有76例病人的数据用于分析。生存分析结果显示miR-181a与乳腺癌的预后相关,miR-181a表达较高的乳腺癌预后较差。进一步分析显示ER阳性乳腺癌中miR-181a对生存有很好的预测作用,具有统计学意义。多因素COX回归分析表明miR-181a、内分泌治疗方式、肿瘤大小和淋巴结转移与乳腺癌预后相关,具有统计学意义。提示miR-181a是乳腺癌预后独立预测因子。双变量相关性分析显示miR-181a表达与年龄、ER、月经状态呈负相关性,而与其他因素无明显相关性,并且统计学有显著意义。结论：体外实验表明miR-181a在他莫昔芬耐药的乳腺癌细胞中表达明显升高。降低乳腺癌细胞的miR-181a表达后,对他莫昔芬的敏感性明显升高。进一步研究显示miR-181a表达受ER表达的影响；miR-181a可以促进mTOR的表达,但对ER, Her-2和PI3K表达无明显影响。临床乳腺癌标本检测发现miR181a的表达水平与乳腺癌患者的预后呈负相关,是ER阳性的乳腺癌预后独立的负性预测因子；本研究数据中miR-181a的表达水平与年龄、ER、月经状态呈负相关性。因此,miR-181a可以作为预测ER阳性乳腺癌预后的潜在指标。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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