MicroRNA455-3p调节三阴性乳腺癌侵袭转移的机制研究

发布时间：2018-01-14 12:27

本文关键词：MicroRNA455-3p调节三阴性乳腺癌侵袭转移的机制研究　出处：《山东大学》2016年硕士论文　论文类型：学位论文

更多相关文章： miR-455-3p 三阴性乳腺癌 浸润转移 EI24

【摘要】：研究背景乳腺癌是一种女性常见的恶性肿瘤,是当前威胁女性生命和健康的主要原因之一。近年来由于经济的飞速发展、生活环境和生活方式的改变以及多方面竞争造成的压力等因素的影响,乳腺癌发病率呈逐年增高的趋势。作为一种异质性疾病,乳腺癌在形态、临床表现及对治疗的敏感性上均有所差异。三阴性乳腺癌(triple negative breast cancer, TNBC)是乳腺癌分子亚型分型中的一种特殊类型,因其分子表型缺乏雌激素受体(estrogen receptor, ER)、孕激素受体(progestrone receptor, PR)和人表皮生长因子受体-2(human epidermal growth factor 2, HER-2)的表达而得名。TNBC约占所有乳腺癌的15-20%,与其他类型的乳腺癌相比,好发于相对年轻的妇女,其细胞分化程度更差,侵袭性更强,更易出现早期复发和远处转移,因此临床预后差。目前TNBC在临床上的主要治疗方法是外科手术和辅助性化疗,但因缺乏激素受体及HER-2等治疗靶点,尚无针对性的有效治疗方案,因此TNBC患者的预后仍不理想。近年来TNBC发生的分子机制已成为众多学者关注和研究的热点。MicroRNA(简称miRNA)是一类含有18～22个核苷酸的内源性的非编码的RNA分子,它广泛存在于真核生物中,是一类重要的基因表达调控因子,在细胞的增殖、发育、分化及凋亡中发挥着重要作用。miRNA可以通过和mRNA的31-UTR区特异性结合抑制靶基因的转录或诱导mRNA的降解,在生物体内发挥癌基因或抑制癌基因的作用。根据生物信息学分析,人类基因的92%受miRNA的调节,现有证据表明miRNA的异常调节参与乳腺癌的发生发展。例如miR-10b在转移性乳腺癌中表达上调,通过负性调控HOXD10促进乳腺癌的转移和浸润,乳腺癌中miR-106b的高表达,预示着病人复发的风险较大,而miR-206在Noth3抑制乳腺癌细胞的增殖、浸润和转移,但目前为止在TNBC中确认功能的miRNA却少之又少。作为p53的一个反应性凋亡前体因子,E124在抑制细胞生长以及自噬激活的过程中起到了重要的作用。而且,EI24的低表达与EMT的诱导和肿瘤的进展存在密切的联系：EI24的缺失引起的依托泊苷抵抗,表明其可能成为一个指示化疗有效性的潜在因子。Jung-Min的研究表明在非小细胞肺癌中降低的EI24表达可以通过IGF-1R信号通路显著提高癌细胞对吉非替尼的抵抗能力；而在食管鳞状细胞癌中,miR-483-3p通过靶向结合EI24基因从而促进了肿瘤的进展,表明miR与EI24之间存在密切的关联。但是,目前尚没有EI24在TNBC中的研究报道。目的通过检测TNBC组织及细胞中microRNA的差异性表达情况,确定候选microRNA;进一步验证其作用及调控的靶基因,探讨其影响TNBC侵袭转移的机制。实验方法(1) miRNA表达谱检测：收集TNBC以及激素受体阳性浸润性导管癌(ER、PR表达均为阳性,HER-2表达呈阴性)石蜡包埋组织各3例,运用miRCURY LNA Array系统对miRNA表达谱进行分析。miRNA表达谱的检测委托联川生物技术公司进行。(2)根据芯片检测结果,筛选出3个在TNBC及对照组中具有差异性表达且有统计学意义的候选miRNA,通过qRT-PCR技术检测117例TNBC和42例激素受体阳性浸润性导管癌组织中3个候选miRNA的表达情况,选取差异性最显著的miRNA进行后续实验。(3)细胞功能学实验,对三阴性乳腺癌细胞株MDA-MB-231和MDA-MB-468体外转染miR-455-3p mimics、negative control、miR-455-3p inhibitor及inhibitor negative control,随后进行MTS和Transwell实验,观察体外其对三阴性乳腺癌细胞增殖、浸润和迁移的影响。(4)生物信息学预测miR-455-3p靶基因,结合芯片结果及相关文献,选定LTBR, EI24, SMAD2作为miR-455-3p可能的靶基因。(5)通过双荧光素酶报告基因实验和Western blot实验,验证miR-455-3p调控的靶基因。结果(1)根据芯片结果分析,筛选出3个候选miRNAs,分别是miR-455-3p, miR-425-5p, miR-196a-5p,其中miR-455-3p和miR-196a-5p在TNBC中显著上调,而miR-425-5p表达呈下调。提取117例三阴性乳腺癌组织和42例浸润性导管癌组织的miRNA,通过qRT-PCR实验检测,结果发现miR-455-3p在三阴性乳腺癌组织中表达上调10倍以上；随后在乳腺癌细胞MCF-7、MDA-MB-231和MDA-MB-468中进行验证,结果显示miR-455-3p在三阴性乳腺癌细胞中表达明显上调,差异具有统计学意义(P0.05)。(2) Transwell细胞实验结果：细胞转染miR-455-3p mimic后迁移和浸润能力明显增强；而转染miR-455-3p inhibitor组的细胞迁移和浸润能力比对照组明显降低,这提示miR-455-3p可以促进三阴性乳腺癌细胞的迁移和浸润。(3)MTS实验结果显示：转染miR-455-3p mimic后提高了细胞的增殖能力；相反,转染miR-455-3p inhibitor后抑制了细胞miR-455-3p的表达,与对照组相比(P0.05),细胞增殖能力减弱,说明miR-455-3p对三阴性乳腺癌细胞的生长和增殖有促进作用。(4)EI24是miR-455-3p的直接靶基因：将EI24荧光素酶载体和miR-455-3p mimic共转染到MDA-MB-231和MDA-MB-468细胞中,miR-455-3p可以直接和EI24的3＇UTR区结合,在转录水平影响EI24的表达,荧光素酶报告基因分析显示,EI24是miR-455-3p的直接靶基因；Western blot实验结果显示,在EI24低表达的三阴性乳腺癌细胞系MDA-MB-231和MDA-MB-468以及EI24高表达的乳腺癌细胞MCF-7中过表达miR-455-3p,EI24蛋白表达均明显降低(P0.05)。进一步验证了EI24是miR-455-3p调控的靶基因。(5)在三阴性乳腺癌细胞中干扰掉EI24后能促进细胞的迁移和浸润,与miR-455-3p过表达对三阴性乳腺癌细胞的影响相似,这提示EI24受miR-455-3p的调控且miR-455-3p能促进三阴性乳腺癌细胞的迁移和浸润。结论miR-455-3p在三阴性乳腺癌组织和三阴性乳腺癌细胞中均显著性上调,发挥着癌基因的作用,体外细胞学实验证实miR-455-3p对三阴性乳腺癌细胞的增殖、浸润和转移起促进作用,其是通过调控EI24促进三阴性乳腺癌的发生发展。
[Abstract]:Background: breast cancer is a common malignant tumor in women, is one of the main causes of threat to women's life and health. In recent years due to the rapid development of economy, the living environment and lifestyle changes and many competitive pressures and other factors, the incidence of breast cancer is increasing year by year. A heterogeneous disease, breast cancer in morphology, clinical manifestations and sensitivity to treatment are different. Three negative breast cancer (triple negative breast cancer, TNBC) is a special type of breast cancer molecular subtypes in the molecular phenotype because of the lack of estrogen receptor (estrogen, receptor, ER) progesterone receptor (progestrone receptor, PR) and human epidermal growth factor receptor -2 (human epidermal growth factor 2, HER-2) expression named approximately.TNBC of all breast cancer 15-20%, and other types of Breast cancer compared to occur in relatively young women, the degree of cell differentiation is even worse, more aggressive, more prone to early recurrence and metastasis, so the prognosis is poor. The main treatment method in clinical TNBC is surgery and adjuvant chemotherapy, but due to lack of hormone receptors and HER-2 therapy there is no point, effective treatment for the patients with TNBC, so the prognosis is still not ideal. The molecular mechanism of TNBC in recent years has become the focus of attention of many scholars and research.MicroRNA (miRNA) is a class containing 18 to 22 nucleotides of endogenous non encoding RNA molecules, it widely exists in eukaryotic organisms, is one of the important factors to regulate the expression of genes in development, cell proliferation, differentiation and apoptosis plays an important role in the.MiRNA 31-UTR region specific through the combination of mRNA and transcription inhibition of target gene or inducing The degradation of mRNA, an oncogene or suppressor role in the organism. According to bioinformatics analysis, the regulation of human gene 92% by miRNA, the existing evidence suggests that abnormal regulation involved in the occurrence and development of breast cancer miRNA. For example the expression of miR-10b was up-regulated in metastatic breast cancer, through the negative regulation of HOXD10 to promote breast cancer metastasis and invasion, high expression of miR-106b in breast cancer, indicates that the greater risk of relapse, miR-206 inhibited the proliferation of breast cancer cells in Noth3, invasion and metastasis, but so far in TNBC confirm the function of miRNA is less and less. As a p53 reaction of apoptosis before the body factor, E124 plays an important role in the process of cell growth inhibition and activation of autophagy. Moreover, there is a close relationship in low expression of EMT and EI24 induced by tumor: the loss of EI24 induced by etoposide In resistance, indicating that it might become a research indicating the effectiveness of chemotherapy potential factor.Jung-Min showed a decrease in non small cell lung cancer EI24 expression through IGF-1R signaling pathway significantly increased cancer cells to gefitinib resistance; in esophageal squamous cell carcinoma, miR-483-3p by targeting to EI24 gene to promote the progress of the tumor shows that there is close correlation between miR and EI24. However, there are no reports on EI24 in TNBC. Through the differential expression of microRNA TNBC detected the tissues and cells, to determine the candidate target gene microRNA; to further verify its effect and regulation, to explore the mechanism of the effect of TNBC the invasion and metastasis of experimental methods. (1) the expression of miRNA and TNBC spectrum detection: collect hormone receptor positive invasive ductal carcinoma (ER, PR expression was positive, HER-2 negative expression) Shi Labao Embedded tissues of the 3 cases, the use of miRCURY LNA Array expression spectrum analysis to detect the expression of.MiRNA by spectrum Lianchuan Biotech Corp of miRNA. (2) according to the result of the microarray, we selected 3 in TNBC and control group with different expression and there was statistical significance when miRNA expression by 3 the candidate miRNA qRT-PCR detection of 117 cases of TNBC and 42 cases of hormone receptor positive invasive ductal carcinoma, select the most significant difference miRNA for subsequent experiments. (3) experimental study on cell function, three negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 in vitro transfection of miR-455-3p mimics, negative control, miR-455-3p inhibitor and inhibitor negative control, followed by MTS and Transwell in vitro experiment, the proliferation of three negative breast cancer cells, effects of infiltration and migration. (4) bioinformatics prediction based on miR-455-3p target Because, according to the microarray results and related literature, selected LTBR, EI24, SMAD2 as the target gene of miR-455-3p. (5) by dual luciferase reporter assay and Western blot experiments, verify the target gene regulated by miR-455-3p. Results (1) according to the chip results, we selected 3 candidate miRNAs, respectively miR-455-3p, miR-425-5p miR-196a-5p, miR-455-3p and miR-196a-5p, which in TNBC was significantly increased, while the expression of miR-425-5p was down regulated. From 117 cases of three breast cancer tissues and 42 cases of invasive ductal carcinoma of the miRNA, through the qRT-PCR test, the results showed that miR-455-3p up-regulated more than 10 times in the three breast cancer tissues; then in breast cancer cells MCF-7, confirmed by MDA-MB-231 and MDA-MB-468, the results showed that the expression of miR-455-3p is upregulated in three negative breast cancer cells, the difference was statistically significant (P0.05). (2) Transw The results of ell cell: cells transfected with miR-455-3p mimic significantly enhanced the ability of migration and invasion; migration of transfected miR-455-3p cells in the inhibitor group and the invasion ability was significantly lower than in the control group, suggesting that miR-455-3p can promote the migration of three negative breast cancer cells and infiltration of MTS. (3) experimental results showed that the transfection of miR-455-3p cells increased after mimic the proliferation ability; instead, after transfection of miR-455-3p inhibitor inhibited the expression of miR-455-3p cells, compared with the control group (P0.05), reduced cell proliferation and miR-455-3p on growth and proliferation of three negative breast cancer cells promoted. (4) EI24 is a direct target gene of miR-455-3p: the EI24 luciferase reporter vector and miR-455-3p mimic and MDA-MB-468 were transfected into MDA-MB-231 cells, with 3 'UTR miR-455-3p can be directly and EI24, at the transcriptional level affect the expression of EI24 Analysis and luciferase reporter gene showed that EI24 is a direct target gene of miR-455-3p Western blot; the experimental results show that miR-455-3p overexpression in breast cancer cells with high expression of MCF-7 in the low expression of EI24 three negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, EI24, EI24 protein expression decreased significantly (P0.05). EI24 is further verified the target genes of miR-455-3p. (5) in three negative breast cancer cells interfered after EI24 can promote cell migration and invasion, and the effect of miR-455-3p overexpression on three negative breast cancer cells, showed that EI24 regulated by miR-455-3p and miR-455-3p can promote the migration of three negative breast cancer cells and infiltration. Conclusion miR-455-3p in three breast cancer tissues and three negative breast cancer cells were significantly up-regulated, play the role of oncogenes, the in vitro experiments showed that miR-455-3p of three yin The proliferation, infiltration and metastasis of sexual breast cancer cells promote the development of three negative breast cancer by regulating EI24.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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