MiRNA-335在三阴性乳腺癌细胞株中的功能探究及其相关靶基因分析

发布时间：2018-06-05 12:33

本文选题：乳腺癌 + 三阴性乳腺癌　；参考：《南京医科大学》2017年博士论文

【摘要】：乳腺癌是当今世界威胁女性健康的主要疾病之一,发病率在世界范围内为恶性肿瘤的第2位,并且仍呈上升趋势。乳腺癌的病理分型对于诊断、治疗、判断预后相当重要,是临床上较成熟的风险评估指标。但近年来,随着乳腺癌的分子生物医学逐渐发展,乳腺癌的分子分型也逐渐发展为临床诊疗的重要组成部分。其中,乳腺癌细胞表面ER、PR、HER-2受体均为阴性的一类乳腺癌称之为三阴性乳腺癌(TNBC),该类乳腺癌具有发病年龄早、易局部复发、易远处转移、预后差等临床特点,并且对于内分泌治疗及靶向治疗均不敏感。因此,越来越多的研究关注于该类型乳腺癌,希望找到对其有效的治疗方法。微小RNA(microRNAs)为内源性非编码单链小RNA,广泛存在于真核细胞中,具有高度保守性,可通过作用于相应的靶基因抑制其蛋白表达来调控细胞的生长、增殖、分化、凋亡、代谢等,与疾病发生密切相关。本研究中,通过查阅文献,我们得到在乳腺癌组织中高表达的miR-335,为了了解miR-335在三阴性乳腺癌细胞中的功能作用及可能的调节机制,本研究从以下两个方面进行实验和探索。第一部分miR-335在MDA-MB-231细胞株和HCC-1937细胞株中的功能研究目的探索miR-335对三阴性乳腺癌细胞株MDA-MB-231和HCC-1937的增殖、迁移能力、侵袭能力的影响。方法运用MTT实验、单克隆形成实验来分析miR-335对细胞增殖能力的影响,通过划痕实验分析对细胞迁移能力的影响,通过Transwell小室实验来分析miR-335对细胞侵袭能力的影响。结果在MDA-MB-231和HCC-1937细胞株中,miR-335上调可抑制细胞的增殖、迁移、侵袭能力,而miR-335表达下调时结果相反。结论在MDA-MB-231和HCC-1937细胞株中,miR-335起到抑癌基因的作用,故其可能作为治疗TNBC的一个候选靶点。第二部分miR-335的靶基因及其机制研究目的分析miR-335可能的靶基因,并初步推测其可能的作用机制。方法通过miR-walk、miR-base、Targetscan等软件寻找miR-335可能的靶基因,运用双荧光素酶报告系统实验进行验证,然后运用Western Blot实验推测miR-335的作用机理。结果双荧光素酶报告实验证实ROCK1作为miR-335的靶点成立,其为miR-335的直接靶基因之一。Western Blot实验结果显示miR-335上调后ROCK1蛋白表达减少;使用miR-335 inhibitor下调miR-335可得到相反结果,进一步证实上述结果。结论在TNBC中,miR-335通过靶向ROCK1来抑制乳腺癌MDA-MB-231细胞株和HCC-1937细胞株的增殖、迁移及侵袭能力。
[Abstract]:Breast cancer is one of the major diseases threatening the health of women in the world. The pathological classification of breast cancer is very important for diagnosis, treatment and prognosis. But in recent years, with the development of molecular biomedicine of breast cancer, molecular classification of breast cancer has gradually developed into an important part of clinical diagnosis and treatment. Among them, the type of breast cancer with negative ERP PR-HER-2 receptors on the surface of breast cancer cells is called tri-negative breast cancer, which is characterized by early onset age, easy local recurrence, easy distant metastasis, poor prognosis and so on. And not sensitive to endocrine therapy and targeted therapy. Therefore, more and more studies focus on this type of breast cancer, hoping to find effective treatment. MicroRNAs are endogenous, non-coding, single-stranded RNAs, widely present in eukaryotic cells and highly conserved to regulate cell growth, proliferation, differentiation, apoptosis, metabolism and so on by acting on target genes to inhibit their protein expression. Is closely related to disease. In this study, we obtained the highly expressed miR-335 in breast cancer tissues. In order to understand the function of miR-335 in triple-negative breast cancer cells and its possible regulatory mechanism, we conducted experiments and exploration in the following two aspects. The function of miR-335 in MDA-MB-231 cell line and HCC-1937 cell line objective to explore the effects of miR-335 on the proliferation, migration and invasion of three negative breast cancer cell lines MDA-MB-231 and HCC-1937. Methods the effect of miR-335 on cell proliferation was analyzed by MTT assay and monoclonal formation assay. The effect of miR-335 on cell migration was analyzed by scratch test. The effect of miR-335 on cell invasion was analyzed by Transwell chamber test. Results in MDA-MB-231 and HCC-1937 cell lines, upregulation of miR-335 inhibited the proliferation, migration and invasion of MDA-MB-231 and HCC-1937 cells, but the expression of miR-335 was down-regulated. Conclusion in MDA-MB-231 and HCC-1937 cell lines, miR-335 plays an important role in tumor suppressor gene, so it may be a candidate target for the treatment of TNBC. The second part is the target gene of miR-335 and its mechanism objective to analyze the possible target gene of miR-335 and to speculate its possible mechanism of action. Methods the possible target gene of miR-335 was found by using miR-walkmiR-base Targetscan software, and verified by double-luciferase reporting system, and then the mechanism of miR-335 was deduced by Western blot. Results the double luciferase report experiment confirmed that Rock1 was the target of miR-335. Western blot showed that the expression of ROCK1 decreased after miR-335 upregulation, and the reverse result could be obtained by using miR-335 inhibitor to down-regulate miR-335. The above results are further confirmed. Conclusion the proliferation, migration and invasion of breast cancer cell line MDA-MB-231 and HCC-1937 were inhibited by targeting ROCK1 in TNBC.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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