miR-223-3p及miR-150-5p在乳腺癌中靶向STIM1的功能研究

发布时间：2018-05-27 16:44

  本文选题：乳腺癌 + STIM1　； 参考：《天津医科大学》2016年硕士论文

【摘要】：目的乳腺癌是女性最常见的恶性肿瘤之一,近年来发病率在环球内呈上升的趋向,其发生和转移是一个与多因素相关的复杂过程。经研究发现,STIM1蛋白是构成钙库操纵性钙通道(store-opetated Ca2+channels,SOCs)的主要成分之一,是细胞内钙离子的传感器,可以使细胞外的钙离子进入细胞内,从而参与乳腺癌的侵袭和转移过程。micro RNA是一类由内源基因编码的非编码单链RNA分子,参与生命活动中的重要进程,约调控人类三分之一的基因。有些miRNAs可以通过转录后负性调控致癌性靶基因表达的方式而发挥抑癌功能;相反,有一部分miRNAs可以通过下调抑癌靶基因表达的方式起到癌基因的作用。在前期工作中,我们小组利用免疫组化的方法发现STIM1蛋白在乳腺IDC组织中呈高表达状态,且STIM1高表达是乳腺癌患者预后的一个不良指标。经生物信息学预测miR-223-3p和miR-150-5p有可能直接调控STIM1基因,从而在乳腺癌的疾病进程中发挥作用。目前在乳腺IDC中miR-223-3p和miR-150-5p与STIM1基因的关系及作用机制尚不清楚,因此本课题旨在研究miR-223-3p-STIM1及miR-150-5p-STIM1在乳腺癌中的相互作用以及两个miRNA和STIM1蛋白与乳腺癌临床病理资料和预后的关系,为乳腺癌病人提供新的预后标志物,并且为乳腺癌的综合治疗需找新的靶点。方法本题选取天津市肿瘤医院2007年1月至2009年12月期间的原发性乳腺IDC患者110例以及其中癌和对应癌旁组织的标本60例,通过查阅病历的方式获取患者的临床病理资料,通过电话随访或者门诊电子检查结果获取随访信息。通过构建双荧光素酶报告基因的方法验证STIM1是miR-223-3p和miR-150-5p直接调控的靶基因,此实验重复三次。采用乳腺癌组织切片原位杂交的方法检测miR-223-3p和miR-150-5p在乳腺癌组织及相应的癌旁组织中的表达量,用Kaplan-Meier法分析miR-223-3p、miR-150-5p和STIM1蛋白与乳腺癌患者总生存时间和无病生存时间的相关性,用卡方检验分析miR-223-3p、miR-150-5p和STIM1蛋白与患者临床病理资料的关系。根据前期中我们用免疫组化方法测定的乳腺癌组织中的STIM1蛋白的表达结果,用Spearman相关法分析miR-223-3p和miR-150-5p分别与STIM1蛋白的相关性,P0.05具有统计学差异。结果1.结果显示,在miR-223-3p组中,在MDA-MB-231和MCF-7两个乳腺癌细胞系中转染STIM1-3,UTR-WT后荧光素酶的相对荧光值明显下降,而转染STIM1-3,UTR-MT后荧光素酶的相对荧光值没有明显变化(P0.05)。说明miR-223-3p可以直接与STIM1-3,UTR区结合,从而影响其生物学功能。在MDA-MB-231和MCF-7乳腺癌细胞系中转染STIM1-3‘UTR-WT/miR-150-5p mimic与STIM1-3,UTR-MT/miR-150-5p mimic的相对荧光值差异并没有统计学意义,P值分别为0.754和0.458。2.miR-223-3p在乳腺癌组织中表达较低,高表达水平的乳腺癌患者的预后优于低表达水平的患者(OS:P=0.038;DFS:P=0.196)。在乳腺癌组织中miR-223-3p表达水平与STIM1蛋白的表达水平呈负相关(r=-1.914,P=0.043)。miR-223-3p的表达水平与患者的淋巴结转移(P=0.035)呈显著相关性。3.miR-150-5p在乳腺癌组织中呈高表达,表达较低的乳腺癌患者的预后有优于表达高的患者的趋势,(OS:P=0.734;DFS:P=0.077)。在乳腺癌组织中miR-150-5p表达水平与STIM1蛋白的表达水平不具有显著的相关性(r=0.001,P=0.991)。miR-150-5p表达水平与乳腺肿瘤大小(P=0.035)、TNM病理分期(P=0.003)以及ER状态(P=0.035)具有显著的关联性。结论1.miR-223-3p在乳腺浸润性导管在组织中表达水平较低,低表达的乳腺癌患者更容易出现淋巴结转移,miR-223-3p高表达组的患者较低表达的患者预后好,STIM1是miR-223-3p直接调控的靶基因,有可能成为治疗乳腺癌的新药物。2.在乳腺浸润性导管癌组织中,miR-150-5p表达较高,miR-150-5p低表达的乳腺癌患者有优于高表达的趋势。miR-150-5p表达水平与患者肿瘤大小和乳腺癌TNM分期以及ER相关,提示miR-150-5p在乳腺癌的侵袭和转移中起到重要作用。
[Abstract]:Objective breast cancer is one of the most common malignant tumors in women. In recent years the incidence of the disease is increasing in the world. Its occurrence and metastasis is a complex process related to multiple factors. It is found that STIM1 protein is one of the main components of store-opetated Ca2+ channels (SOCs), which is the intracellular calcium ionization. The subcellular sensor, which can make the extracellular calcium ions into the cell, participates in the invasion and metastasis of breast cancer, and.Micro RNA is a class of non coded single strand RNA molecules encoded by endogenous genes. It participates in important processes in life activities and regulates human 1/3. Some miRNAs can be induced by post transcriptional negative regulation. On the contrary, a part of miRNAs can play the role of Oncogene by down regulating the expression of tumor suppressor gene. In the early work, our group used immunohistochemical method to find the high expression of STIM1 protein in IDC tissues of the breast, and the high expression of STIM1 is the breast cancer. It is predicted that miR-223-3p and miR-150-5p may directly regulate the STIM1 gene and play a role in the process of breast cancer by bioinformatics. The relationship and mechanism of miR-223-3p and miR-150-5p with the STIM1 gene are still unclear in breast IDC. Therefore, this subject is aimed at studying miR-223-3p-STIM1 The interaction of miR-150-5p-STIM1 in breast cancer and the relationship between two miRNA and STIM1 proteins and the clinicopathological data and prognosis of breast cancer provide a new prognostic marker for breast cancer patients and need to find new targets for the comprehensive treatment of breast cancer. Method this problem was selected from Tianjin Tumour Hospital from January 2007 to December 2009. 110 cases of primary mammary IDC and 60 cases of carcinoma and paracancerous tissue were obtained. The clinicopathological data of the patients were obtained by consulting the medical records. The follow-up information was obtained by telephone follow-up or outpatient electronic examination. The method of constructing the double luciferase reporter gene was used to verify that STIM1 was miR-223-3p and miR-150-5p. The target gene was regulated directly. This experiment was repeated three times. The expression of miR-223-3p and miR-150-5p in breast cancer tissues and corresponding para cancerous tissues was detected by breast cancer tissue section in situ hybridization. The total survival time and disease-free survival time of miR-223-3p, miR-150-5p, STIM1 protein and breast cancer patients were analyzed by Kaplan-Meier method. Correlation, the relationship between miR-223-3p, miR-150-5p and STIM1 protein with the patient's clinicopathological data was analyzed with the chi square test. According to the expression of STIM1 protein in the breast cancer tissue that we used in the early stage of immunohistochemistry, the correlation of miR-223-3p and miR-150-5p with STIM1 protein was analyzed by Spearman correlation method, and P0.05 has a series of data. Results 1. results showed that in group miR-223-3p, STIM1-3 was transfected into two breast cancer cell lines in MDA-MB-231 and MCF-7, and the relative fluorescence value of luciferase was obviously decreased after UTR-WT, while STIM1-3 was transfected with STIM1-3, and the relative fluorescence of luciferase was not significantly changed after UTR-MT (P0.05). It indicated that miR-223-3p could be directly associated with STIM1-3, UTR region. In MDA-MB-231 and MCF-7 breast cancer cell lines transfected with STIM1-3 'UTR-WT/miR-150-5p mimic and STIM1-3, the relative fluorescence values of UTR-MT/miR-150-5p mimic are not statistically significant, and P values are 0.754 and 0.458.2.miR-223-3p in breast cancer tissues that express low, high expression levels of breast cancer. The prognosis of patients was better than that of low expression level (OS:P=0.038; DFS:P=0.196). The expression level of miR-223-3p in breast cancer tissues was negatively correlated with the expression level of STIM1 protein (r=-1.914, P=0.043).MiR-223-3p expression level was significantly correlated with lymph node metastasis (P=0.035) in patients with high expression of.3.miR-150-5p in breast cancer tissue. The prognosis of patients with low expression of breast cancer is better than that of high expression patients (OS:P=0.734; DFS:P=0.077). There is no significant correlation between the expression level of miR-150-5p and the expression level of STIM1 protein in breast cancer tissues (r=0.001, P=0.991).MiR-150-5p expression and breast tumor size (P=0.035), TNM pathological staging (P=0.003) ER state (P=0.035) has a significant correlation. Conclusion the expression level of 1.miR-223-3p in mammary gland infiltrating ducts is low. The patients with low expression of breast cancer are more prone to lymph node metastasis, and the patients with miR-223-3p high expression group have better prognosis. STIM1 is the target gene directly regulated by miR-223-3p, and it may become a cure. .2., a new drug for breast cancer, has a high expression of miR-150-5p in invasive ductal carcinoma of the breast. The expression level of breast cancer with low expression of miR-150-5p is superior to that of high expression of.MiR-150-5p, which is associated with the size of the tumor and the TNM staging of the breast cancer and ER, suggesting that miR-150-5p plays an important role in the invasion and metastasis of breast cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9
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本文编号：1943004