miR-29b靶向作用于HER-2对乳腺癌细胞生物学功能的影响

发布时间：2018-06-12 20:12

本文选题：乳腺癌 + miR-29b　；参考：《南昌大学》2016年博士论文

【摘要】：乳腺癌(Breast Cancer)是一种女性恶性肿瘤,对患者身心产生巨大伤害。目前对于造成乳腺癌患者死亡的具体原因机制并不清楚。在肿瘤等多种疾病中,微小RNA(microNRA,miRNA)具有重要的功能,miRNA既可作为抑癌基因也可作为致癌基因,从而在肿瘤发生发展的全过程中发挥不同的作用,同时miRNA自身也可以发生突变或缺失等现象,进而影响相关基因的表达发生异常。对miRNA进行分析,可发现多种miRNA与生命活动关键蛋白和信号通路的调节有关,通过对这些蛋白和通路进行研究,可了解miRNA发挥的作用的具体机制。而乳腺癌的发生发展与转移等过程是由许多miRNA信号分子调控的,通过上述通路调节的研究,我们可以得出miRNA在乳腺癌中的的作用,从而为乳腺癌的前期诊断和后期治疗提供一些新的思路和方法。miR-29b作为miRNA中的成员之一,参与了多种肿瘤细胞的增殖、分化、凋亡、侵袭和转移过程。miR-29b已成为与肿瘤诊断、治疗和预后相关的具有潜在价值的肿瘤标志物。然而,miR-29b在乳腺癌中的作用目前尚未明确,因此,研究miR-29b在乳腺癌发生发展过程中的作用及其作用靶点的探讨十分具有意义。目的:探讨微小RNA-29b(miRNA-29b)在乳腺癌组织和细胞中的表达水平,分析miRNA-29b的表达与乳腺癌患者临床病理参数及预后的相关性,观察miRNA-29b对乳腺癌细胞周期、增殖、凋亡和迁移的影响,生物信息学预测miRNA-29b的靶基因,并初步探讨其可能的作用机制。方法:采用实时荧光定量PCR的方法检测miR-29b在乳腺癌细胞株和乳腺癌患者癌组织中的表达,采用总RNA提取试剂盒提取新鲜冰冻组织及细胞株中总RNA,使用PrimeScript RT试剂盒反转录法合成cDNA,ABI 7500荧光定量PCR仪进行PCR反应;采用统计分析法对乳腺癌癌组织中mi R-29b水平与患者临床病理参数进行相关性分析。CCK8法和细胞划痕实验分别检测过表达mi R-29b后乳腺癌细胞增殖和迁移,流式细胞术检测乳腺癌细胞的细胞周期和凋亡情况。通过TargetScan和miRanda等靶基因预测数据库对miR-29b的靶基因进行预测,分析miR-29b过表达与HER-2表达的相关性,研究miR-29b过表达对HER-2蛋白的影响,Western blotting检测过表达miR-29b的乳腺癌细胞中HER-2的表达,初步验证miR-29b的作用靶点。结果:miR-29b在乳腺癌组织和乳腺癌细胞中的表达显著下调;miR-29b过表达能够抑制乳腺癌细胞的增殖,促进起凋亡,并减弱其迁移和侵袭能力(P0.01);miR-29b与HER-2存在直接靶向负作用关系,乳腺癌患者癌组织中mi R-29b的表达水平与患者年龄和浸润程度之间无显著相关性(P0.05),而与患者乳腺癌组的肿瘤大小和HER-2及淋巴结转移有显著相关性(P0.01),乳腺癌患者的预后与乳腺癌组织中miR-29b的表达有很明显的关系:miR-29b表达越低,患者预后越差(P0.01)
[Abstract]:Breast Cancer (Breast Cancer) is a female malignant tumor that causes great damage to the body and mind of patients. The specific causes of breast cancer deaths are unclear. In many diseases, such as tumor, microRNAs have important function: miRNAs can be used as tumor suppressor gene and oncogene, so they play different roles in the whole process of tumorigenesis and development. At the same time, miRNA itself can be mutated or deleted, thus affecting the abnormal expression of related genes. Through the analysis of miRNA, we can find that many miRNAs are related to the regulation of vital proteins and signaling pathways. Through the study of these proteins and pathways, we can understand the specific mechanism of miRNA's action. However, the development and metastasis of breast cancer are regulated by many miRNA signaling molecules. Through the study of the regulation of these pathways, we can conclude the role of miRNA in breast cancer. As a member of miRNA, miR-29b is involved in the proliferation, differentiation, apoptosis, invasion and metastasis of many kinds of tumor cells. A potentially valuable tumor marker associated with treatment and prognosis. However, the role of miR-29b in breast cancer is not clear, so it is of great significance to study the role and target of miR-29b in the development of breast cancer. Objective: to investigate the expression level of miRNA-29b in breast cancer tissues and cells, analyze the relationship between miRNA-29b expression and clinicopathological parameters and prognosis of breast cancer patients, and observe the effect of miRNA-29b on cell cycle, proliferation, apoptosis and migration of breast cancer. The target gene of miRNA-29b was predicted by bioinformatics and its possible mechanism was discussed. Methods: the expression of miR-29b in breast cancer cell line and breast cancer tissue was detected by real-time fluorescent quantitative PCR. Total RNA extraction kit was used to extract total RNAs from fresh frozen tissues and cell lines. CDNAABI 7500 was synthesized by reverse transcription of primeScript RT kit for PCR reaction. The correlation between the expression of mi R-29b and the clinicopathological parameters of breast cancer tissues was analyzed by statistical analysis. The proliferation and migration of breast cancer cells after overexpression of mi R-29b were detected by CCK8 method and cell scratch assay. Cell cycle and apoptosis of breast cancer cells were detected by flow cytometry. The target gene of miR-29b was predicted by target gene prediction database such as TargetScan and miRanda. The correlation between the overexpression of miR-29b and HER-2 expression was analyzed. The effect of overexpression of miR-29b on HER-2 protein was studied. The expression of HER-2 in breast cancer cells with miR-29b overexpression was detected by Western blotting. The action target of miR-29b was preliminarily verified. Results the overexpression of miR-29b in breast cancer tissues and breast cancer cells significantly down-regulated the expression of miR-29b, which could inhibit the proliferation of breast cancer cells, promote apoptosis, and weaken the migration and invasion ability of human breast cancer cells. There was a direct and negative relationship between the expression of miR-29b and HER-2 in breast cancer tissues and breast cancer cells. There was no significant correlation between the expression of mi R-29b in breast cancer tissue and age and degree of invasion (P 0.05), but a significant correlation with tumor size, HER-2 and lymph node metastasis in patients with breast cancer (P 0.01). The lower the expression of miR-29b, the lower the expression of miR-29b in breast cancer. The worse the prognosis was (P0.01)
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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