整合素连接激酶对乳腺癌细胞增殖的作用及其机制研究

发布时间：2018-06-06 12:54

本文选题：乳腺癌 + 整合素连接激酶　；参考：《天津医科大学》2017年博士论文

【摘要】：目的:乳腺癌是世界范围内女性中最常见的恶性肿瘤之一,其发病率位于女性所有肿瘤中的第一位。尽管乳腺癌主要的治疗策略是外科手术结合放疗和化疗,但近年来一种全新的治疗手段即分子靶向治疗逐步被人们认识并接受,特别是广泛应用于对上述疗法无效的广大患者。然而,由于调控乳腺癌进程的分子机制仍然没有完全阐明,故导致分子靶向治疗缺乏有效的治疗靶点。因此,探索乳腺癌细胞生长的调控机制,将有利于提高乳腺癌分子靶向治疗的效率和为其提供潜在的治疗靶点。肿瘤细胞在侵袭和扩散的过程中,依赖于肿瘤细胞与细胞外基质之间粘附的缺失,而该过程可由整合素所介导。整合素连接激酶(Integrin-linked kinase,ILK)是一种重要的整合素丝氨酸-苏氨酸蛋白激酶,其广泛表达于人体各个组织中。先前的研究已经表明,在多种肿瘤中均观察到整合素连接激酶的表达上调,并且整合素连接激酶还参与调节肿瘤细胞的增殖、细胞周期进程、侵袭和迁移、细胞活性及肿瘤血管的发生等多种生理功能。在乳腺癌中,活化的整合素连接激酶可通过结合LIMD 2而促进乳腺癌细胞的运动和转移能力。然而,目前还未见关于整合素连接激酶参与乳腺癌细胞增殖的作用及其机制的报道。本研究利用基因转染和RNAi来研究过表达及沉默整合素连接激酶基因对乳腺癌细胞增殖的作用及其机制,该研究将有助于人们进一步认识乳腺癌的发生机制,并为乳腺癌标志物和靶点的确立提供重要的参考依据。方法:1、利用基因转染的方法建立稳定转染ILK的MCF-7乳腺癌细胞株;WST-1、BrdU掺入和LDH释放实验研究过表达ILK对MCF-7细胞增殖、活力及凋亡的影响;Western blot实验检测MCF-7细胞过表达ILK对Akt、p-Akt1(Thr308)和PCNA基因蛋白表达的影响。2、利用RNAi方法建立低表达ILK的MDA-MB-231乳腺癌细胞株;WST-1、BrdU掺入和LDH释放实验研究低表达ILK对MDA-MB-231细胞增殖、活力及凋亡的影响;Western blot实验检测MDA-MB-231细胞低表达ILK对Akt、p-Akt1(Thr308)和PCNA基因蛋白表达的影响。3、采用Akt抑制剂LY294002来抑制过表达ILK的MCF-7细胞中p-Akt1(Thr308)的表达;WST-1、BrdU掺入和LDH释放实验研究在过表达ILK的MCF-7细胞中抑制PI3K/Akt通路对细胞增殖、活力及凋亡的影响;Western blot实验检测封闭PI3K/Akt通路对过表达ILK的MCF-7细胞PCNA基因蛋白表达的影响。4、通过瞬时转染HA-AKT使低表达ILK的MDA-MB-231细胞中PI3K/Akt通路被活化;WST-1、BrdU掺入和LDH释放实验研究活化PI3K/Akt通路对低表达ILK的MDA-MB-231细胞增殖、活力及凋亡的影响;Western blot实验检测活化PI3K/Akt通路对低表达ILK的MDA-MB-231细胞PCNA基因蛋白表达的影响。结果:1、WST-1、BrdU掺入和LDH释放实验表明过表达ILK可以促进MCF-7细胞的增殖和活力、抑制其凋亡;Western blot实验表明过表达ILK可上调MCF-7细胞中p-Akt1(Thr308)和PCNA基因的蛋白表达,但Akt蛋白的表达量没有受到ILK基因转染的影响。2、WST-1、BrdU掺入和LDH释放实验表明低表达ILK可以抑制MDA-MB-231细胞的增殖和活力、促进其凋亡;Western blot实验表明MDA-MB-231细胞低表达ILK可下调p-Akt1(Thr308)和PCNA基因的蛋白表达,但Akt蛋白的表达量没有受到ILK基因沉默的影响。3、10μM的LY294002可显著抑制过表达ILK的MCF-7细胞中p-Akt1(Thr308)的表达,但Akt的表达不受影响,表明LY294002可有效地阻断MCF-7细胞中PI3K/Akt通路;WST-1、BrdU掺入和LDH释放实验表明封闭PI3K/Akt通路可抑制过表达ILK的MCF-7细胞的增殖和活力,促进其凋亡。另外,Western blot实验表明封闭PI3K/Akt通路可下调过表达ILK的MCF-7细胞PCNA蛋白的表达。4、瞬时转染HA-AKT可显著上调低表达ILK的MDA-MB-231细胞中p-Akt1(Thr308)的表达,但Akt的表达不受影响,表明瞬时转染HA-AKT可有效活化MDA-MB-231细胞中PI3K/Akt通路;WST-1、BrdU掺入和LDH释放实验表明活化PI3K/Akt通路可促进低表达ILK的MDA-MB-231细胞的增殖和活力,抑制其凋亡。另外,Western blot实验表明活化PI3K/Akt通路可上调低表达ILK的MDA-MB-231细胞PCNA蛋白的表达。结论:本研究论证了ILK可通过活化PI3K/Akt通路而促进乳腺癌细胞的增殖和活力,并抑制其凋亡,这表明ILK作为致癌基因在乳腺癌的发展中扮演着重要的作用。该研究成果将有助于人们对乳腺癌发生机制的进一步认识,并可为乳腺癌的分子靶向治疗提供有效的潜在靶点。
[Abstract]:Objective: breast cancer is one of the most common malignant tumors in women worldwide. The incidence of breast cancer is the first in all women's tumors. Although the main treatment strategy for breast cancer is surgery combined with radiotherapy and chemotherapy, a new treatment, molecular targeting therapy, is gradually recognized and accepted in recent years, especially in molecular targeting therapy. However, the molecular mechanism of the regulation of breast cancer is still not fully elucidated, which leads to the lack of effective therapeutic targets for molecular targeting therapy. Therefore, the study of the regulatory mechanism of the growth of breast cancer cells will be beneficial to the efficiency of molecular targeting therapy for breast cancer and to improve the efficiency of molecular targeting therapy for breast cancer. For potential therapeutic targets, tumor cells are dependent on the loss of adhesion between tumor cells and extracellular matrix during invasion and diffusion, and this process can be mediated by integrin. Integrin linked kinase (Integrin-linked kinase, ILK) is an important integrin serine - threonine protein kinase, which is widely expressed in human body In various tissues, previous studies have shown that the expression of integrin linked kinase is up-regulated in a variety of tumors, and integrin linked kinase is also involved in regulating the proliferation of tumor cells, cell cycle processes, invasion and migration, cell activity and the development of tumor vessels. In breast cancer, activation integration Hormone linked kinase can promote the movement and metastasis of breast cancer cells by combining LIMD 2. However, there is no report on the role and mechanism of integrin linked kinase in the proliferation of breast cancer cells. This study uses gene transfection and RNAi to study the expression and silencing of integrin linked kinase gene to breast cancer cells The effect and mechanism of proliferation will help people to further understand the mechanism of breast cancer and provide important reference for the establishment of markers and targets of breast cancer. Methods: 1, a MCF-7 breast cancer cell line stably transfected with ILK is established by gene transfection; WST-1, BrdU incorporation and LDH release experiment study the expression of I The effect of LK on the proliferation, vitality and apoptosis of MCF-7 cells. Western blot test detected the effect of MCF-7 cells over expression of ILK on the expression of Akt, p-Akt1 (Thr308) and PCNA gene protein. The effect of Western blot on the expression of Akt, p-Akt1 (Thr308) and PCNA gene protein by the low expression of ILK in MDA-MB-231 cells was detected by the Akt inhibitor LY294002. The effect of PI3K/Akt pathway on cell proliferation, activity and apoptosis; Western blot test detected the effect of closed PI3K/Akt pathway on the expression of PCNA gene protein in MCF-7 cells overexpressing ILK,.4 was activated by transient transfection of HA-AKT to the PI3K/Akt pathway in MDA-MB-231 cells with low expression of ILK. The effect of K/Akt pathway on the proliferation, activity and apoptosis of MDA-MB-231 cells with low expression of ILK. Western blot test was used to detect the effect of activated PI3K/Akt pathway on the expression of PCNA gene protein in MDA-MB-231 cells with low expression of ILK. Results: 1, WST-1, BrdU incorporation and LDH release experiments showed that overexpression could promote the proliferation and vitality of the cells and inhibit it. Apoptosis, Western blot test showed that overexpression of ILK could up regulate the protein expression of p-Akt1 (Thr308) and PCNA genes in MCF-7 cells, but the expression of Akt protein was not affected by ILK gene transfection,.2, WST-1. The results showed that low expression of ILK in MDA-MB-231 cells could down regulate the protein expression of p-Akt1 (Thr308) and PCNA gene, but the expression of Akt protein was not affected by ILK gene silencing, and LY294002 could inhibit the expression of ILK MCF-7 cells that overexpressed ILK, but the expression was not affected. It indicated that the Akt could be effectively blocked. The PI3K/Akt pathway in MCF-7 cells, WST-1, BrdU incorporation and LDH release experiments showed that closed PI3K/Akt pathway could inhibit the proliferation and vitality of MCF-7 cells overexpressing ILK and promote its apoptosis. The expression of p-Akt1 (Thr308) in MDA-MB-231 cells expressed in ILK was lowered, but the expression of Akt was not affected, which indicated that transient transfection of HA-AKT could effectively activate PI3K/Akt pathway in MDA-MB-231 cells; WST-1, BrdU incorporation and LDH release experiment showed that activation PI3K/Akt pathway could promote the proliferation and vitality of low expression cells and inhibit its apoptosis. In addition, the Western blot experiment showed that activated PI3K/Akt pathway could increase the expression of PCNA protein in MDA-MB-231 cells with low expression of ILK. Conclusion: This study demonstrated that ILK can promote the proliferation and activity of breast cancer cells by activating PI3K/Akt pathway and inhibit its apoptosis, which indicates that ILK as a oncogene plays an important role in the development of breast cancer. The results will help people to further understand the mechanism of breast cancer and provide a potential target for molecular targeting therapy for breast cancer.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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