DVL3和细胞外因子β-catenin的表达与乳腺癌细胞发展的相关性及其机制研究

发布时间：2018-01-29 18:13

本文关键词： AMPK DVL3 β-catenin 二甲双胍细胞生长乳腺癌　出处：《南昌大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:AMPK(Adenosine 5’-monophosphate-activated protein kinase)即AMP依赖性蛋白激酶是一种丝氨酸/苏氨酸蛋白激酶,是调节能量代谢的关键分子,是机体中能源系统的主要监管机构,在细胞或组织中可以通过调节下游信号分子DVL3和β-catenin蛋白调控细胞新陈代谢和保护其免受各种外界压力的功能。AMPK激活剂能够通过对肿瘤细胞的生长进行抑制进而达到治疗疾病的效果。但是,对于AMPK如何活化和如何在癌症中抑制癌细胞增殖的分子机制到目前仍然并不十分清楚。我们通过研究AMPK激活剂对乳腺癌细胞和组织的影响,以期阐明其作用的分子机制,为将来用于临床治疗提供可靠的理论依据。方法:收集并用Western Blot和免疫组化法分析40例乳腺癌患者组织中AMP依赖性蛋白激酶下游分子DVL3和β-catenin的蛋白质表达水平与乳腺癌细胞病理特征,预后以及不同分子亚型之间的相关性。利用Western Blot法检测了加载有DVL3质粒或者shRNA-DVL3的MCF-7、MDA-MB-231、T-47D乳腺癌细胞和MCF-10乳腺细胞的DVL3表达和细胞周期的变化。利用AMPK的激活剂二甲双胍处理乳腺癌细胞通过体外划痕实验,Transwell法以及细胞克隆实验检测乳腺癌细胞的发展。构建了乳腺癌移植瘤的模式小鼠结合HE染色和Western Blot法检测了DVL3和β-catenin的表达水平。结果:乳腺癌组织中存在DVL3的过表达,并且与分型、分化、淋巴结转移和分期相关,DVL3在淋巴结转移灶的表达明显高于原发灶。DVL3过表达与异常表达跟乳腺癌的不良预后正相关,并可作为影响乳腺癌预后的独立危险因素。DVL3能够与Wnt/β-catenin信号通路作用,显著调节癌细胞的生长。AMPK激活剂有可能抑制乳腺癌细胞的生长,减少DVL3与Wnt/β-catenin信号的表达。进一步对转染过表达或者抑制DVL3的乳腺癌细胞进行研究,发现DLV3参与了细胞的增殖,迁移和凋亡等过程。AMPK激活剂是通过减少DVL3的表达和降低β-catenin水平来抑制乳腺癌细胞的生长。免疫印迹和免疫组织化学显示DVL3与β-catenin表达水平显著相关。过表达DVL3导致β-catenin表达水平上升和乳腺癌细胞的过度生长,这证实通过Wnt/β-catenin通路激活的DVL3与乳腺癌的发展有关。为了阐明机制,我们在体内和体外使用了AMPK活化剂确认对DVL3和β-catenin是否存在依赖关系。结论:DLV3不仅在乳腺癌中存在着过表达,而且是通过经典的信号通路而发挥促进乳腺癌细胞增殖,迁移和侵袭的能力。AMPK激活剂二甲双胍能够抑制DVL3的表达水平,从而降低β-catenin,c-Myc和CyclinD1蛋白的表达,最终导致了乳腺癌细胞的增殖、迁移和侵袭的明显抑制。本研究结果证实,本论文的研究结果对于临床有较高的应用价值,同时能够提倡使用二甲双胍通过特定分子机制对乳腺癌进行治疗。
[Abstract]:Objective: Adenosine 5 monophosphate-activated protein kinase). AMP dependent protein kinase is a serine / threonine protein kinase. It is the key molecule of regulating energy metabolism and the main regulatory body of energy system in the body. A function in cells or tissues that regulates cell metabolism and protects cells from various external pressures by regulating downstream signaling molecules DVL3 and 尾 -catenin proteins. AMPK activators can be activated by. To inhibit the growth of tumor cells to achieve the effect of disease treatment. The molecular mechanism of how AMPK is activated and how it inhibits the proliferation of cancer cells remains unclear. We study the effects of AMPK activators on breast cancer cells and tissues. In order to elucidate the molecular mechanism of its action. To provide a reliable theoretical basis for future clinical treatment. Methods: collect and use Western. The expression levels of DVL3 and 尾 -catenin downstream molecules of AMP dependent protein kinase and the pathological characteristics of breast cancer cells were analyzed by Blot and immunohistochemistry. Western Blot method was used to detect MCF-7 loaded with DVL3 plasmid or shRNA-DVL3. MDA-MB-231. The changes of DVL3 expression and cell cycle in T-47D breast cancer cells and MCF-10 breast cancer cells. The AMPK activator metformin was used to treat breast cancer cells by in vitro scratch assay. Transwell assay and cell cloning assay were used to detect the development of breast cancer cells. A model mouse model of breast cancer transplantation was constructed with HE staining and Western. The expression levels of DVL3 and 尾 -catenin were detected by Blot. Results: there was overexpression of DVL3 in breast cancer. The expression of DVL3 in lymph node metastasis was significantly higher than that in primary tumor. The abnormal expression of DVL3 was positively correlated with the poor prognosis of breast cancer. DVL3 may play an important role in Wnt / 尾 -catenin signaling pathway. Significantly regulate the growth of cancer cells. AMPK activator may inhibit the growth of breast cancer cells. Reduce the expression of DVL3 and WNT / 尾 -catenin signal. Further study on breast cancer cells transfected with DVL3 or inhibit the expression of DLV3, we found that DLV3 involved in cell proliferation. AMPK activator inhibits the growth of breast cancer cells by reducing the expression of DVL3 and the level of 尾 -catenin during migration and apoptosis. Western blot and immunohistochemistry showed DVL. 3There was a significant correlation between 尾 -catenin expression and 尾 -catenin expression. Overexpression of DVL3 resulted in the increase of 尾 -catenin expression and over-growth of breast cancer cells. This suggests that DVL3 activated through the Wnt- 尾 -catenin pathway is associated with the development of breast cancer, in order to elucidate the mechanism. We used AMPK activator in vivo and in vitro to confirm whether there is a dependence on DVL3 and 尾 -catenin. Conclusion: DLV3 is not only overexpressed in breast cancer. And it is through the classic signal pathway to promote the proliferation, migration and invasion of breast cancer cells. Metformin, an activator of AMPK, can inhibit the expression of DVL3. Thus, the expression of 尾 -cateninine c-Myc and CyclinD1 protein was decreased, and the proliferation, migration and invasion of breast cancer cells were significantly inhibited. The results of this study are of high clinical value and can promote the use of metformin in the treatment of breast cancer through specific molecular mechanisms.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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