RNAi阻低UBE3A后对乳腺癌细胞MDA-MB-231生物学行为的影响及其机制研究

发布时间：2018-04-05 00:36

本文选题：乳腺癌　切入点：UBE3A　出处：《川北医学院》2015年硕士论文

【摘要】：第一部分RNAi阻低UBE3A后对乳腺癌细胞MDA-MB-231生物学行为的影响目的:通过sh RNA及慢病毒载体阻低UBE3A的表达后,观察乳腺癌细胞株MDA-MB-231生物学行为的变化,以探讨UBE3A在乳腺癌细胞中的作用。方法:构建3条干扰UBE3A基因的表达载体UBE3A-sh RNA1、UBE3A-sh RNA2、UBE3A-sh RNA3和1条无关序列作为阴性对照Negative-sh RNA;在脂质体lipo2000介导下转化293T细胞,包装产生慢病毒载体,再将慢病毒载体转染至乳腺癌细胞株MDA-MB-231;荧光定量PCR检测UBE3A的干扰效率,筛选出干扰效率最高的干扰片段;Western blotting检测各组细胞UBE3A蛋白水平上的阻低情况;Transwell小室侵袭实验检测UBE3A阻低后,MDA-MB-231细胞侵袭能力的变化;流式细胞术检测阻低UBE3A后,MDA-MB-231细胞周期情况;CCK-8试剂盒法检测阻低UBE3A后,MDA-MB-231细胞在24h、48h、72h增殖能力变化;对结果采用SPSS 22.0进行统计分析,以P0.05为差异有统计学意。结果:成功构建及筛选出了干扰率最高的慢病毒载体LV-UBE3A-sh RNA;Q-PCR、Western blotting结果显示:相对空白组和阴性对照组,UBE3A阻低组UBE3A基因和蛋白表达均受到抑制,抑制率分别为89.5%、45.3%,差异具有统计学意义(P0.05),空白组和阴性对照组差异无统计学意义(P0.05);CCK-8实验及Transwell小室侵袭实验结果显示:UBE3A阻低组与空白对照组及阴性对照组相比较,MDA-MB-231细胞增殖、侵袭能力均受抑制,差异具有统计学意义(P0.05),空白组与阴性对照组差异无统计学意义(P0.05);流式细胞术结果显示:阻低UBE3A组与空白对照组及阴性对照组MDA-MB-231细胞各周期差异具有统计学意义(P0.05),且阻低UBE3A组主要变化集中在S期,空白组与阴性对照组差异无统计学意义(P0.05)。结论:UBE3A阻低后,MDA-MB-231细胞的增殖能力、细胞周期以及细胞的侵袭能力等生物学行为发生明显变化,提示UBE3A在乳腺癌细胞中扮演着重要角色,其可能是乳腺癌治疗的一个潜在靶标。第二部分阻低UBE3A后对乳腺癌细胞MDA-MB-231生物学行为影响的机制研究目的:通过2-DE为基础的差异蛋白质组学方法观察UBE3A阻低前后乳腺癌细胞株MDA-MB-231蛋白质的表达差异,探讨UBE3A对乳腺癌生物学行为影响的机制。方法:采用2-DE方法对UBE3A阻低前后MDA-MB-231细胞的总蛋白进行分离,PDQuest 7.1软件进行分析获得差异蛋白质,然后使用串联质谱(MALDI-TOF-TOF-MS/MS)对差异蛋白质进行鉴定;采用DAVID Functional Annotation及String在线工具分析差异表达蛋白质的细胞成分、生物学途径、分子功能及其之间的相互关系。结果:2-DE及串联质谱鉴定出阻低UBE3A前后28个差异蛋白质点,其中有4个蛋白质在阻低UBE3A组中相对高表达,24个相对低表达,除去鉴定相同的蛋白质后有24个差异蛋白质,与DAVID数据库有23个相匹配;DAVID Functional Annotation进行GO分类,能够进行生物学途径、细胞成分、分子功能注释分类的蛋白质分别为23(100%)、20(87.7%)和23(100%),而在泛素-蛋白酶体系统及糖代谢方面分类比较明确;String分析显示这些差异蛋白中有23个存在直接或间接的联系。结论:23个差异蛋白质之间存在着直接或间接的联系,特别在泛素-蛋白酶体系统功能及糖代谢功能方面发生变化,这可能是阻低UBE3A后乳腺癌细胞生物学行为改变的原因。
[Abstract]:The first part influence on the biological behavior of human breast cancer cell line MDA-MB-231 RNAi UBE3A low resistance Objective: the expression of SH after RNA and lentiviral vector resistance is low after UBE3A, to observe the change of biological behavior of breast cancer cell line MDA-MB-231, to investigate the role of UBE3A in breast cancer cells. Methods: the construction of 3 interference UBE3A gene expression UBE3A-sh RNA2, carrier UBE3A-sh RNA1, UBE3A-sh RNA3 and 1 unrelated sequence as negative control Negative-sh RNA; transformation of 293T cells mediated by liposome lipo2000, packaged lentivirus vector, then lentiviral vector transfected into human breast cancer cell line MDA-MB-231; interference efficiency of fluorescence quantitative PCR detection of UBE3A, screening out the interference fragment the highest interference efficiency; Western blotting cells were detected with UBE3A protein levels on the low resistance; Transwell chamber invasion assay UBE3A low resistance after the invasion of MDA-MB-231 cells The ability to change; flow cytometry was used to detect the resistance of low UBE3A, MDA-MB-231 cell cycle; CCK-8 kit method was used to detect the resistance of low UBE3A, MDA-MB-231 cells in 24h, 48h, 72h changes in proliferation ability; using SPSS 22 statistical analysis of the results by P0.05 were statistically. Results: the successful construction and selected interference rate of lentiviral vector of LV-UBE3A-sh RNA Q-PCR Western blotting, the highest; the results showed that: the relative control group and negative control group, low UBE3A group expression of UBE3A gene and protein resistance were inhibited, the inhibition rates were 89.5%, 45.3%, the difference was statistically significant (P0.05), blank control group and negative control group no statistical significance (P0.05); CCK-8 assay and Transwell invasion assay showed that: UBE3A low resistance group and blank control group and negative control group compared to MDA-MB-231 cell proliferation and invasion were inhibited, with differences There was statistical significance (P0.05), the control group and negative control group had no significant difference (P0.05); flow cytometry results showed that the resistance of the low UBE3A group and blank control group and negative control group MDA-MB-231 cells in each cycle. The difference was statistically significant (P0.05), and the resistance of the low UBE3A group mainly concentrated in the change of S phase. The blank control group and negative control group had no significant difference (P0.05). Conclusion: UBE3A resistance is low, the proliferation of MDA-MB-231 cells, significant changes in the biological behavior of cell cycle and cell invasion, suggesting that UBE3A plays an important role in breast cancer cells, which may be a potential target for the treatment of breast cancer the purpose of research on the mechanism of resistance. The second part is low after UBE3A on the biological behavior of breast cancer cell MDA-MB-231 effect: by the difference of 2-DE based proteomics method to observe the UBE3A low resistance before and after breast cancer cell line MDA- The differential expression of MB-231 protein, investigate the mechanism of effect of UBE3A on the biological behavior of breast cancer. Methods: using the 2-DE method of total protein of UBE3A low resistance MDA-MB-231 cells before and after the separation, PDQuest 7.1 was used to analyze the obtained proteins, then using tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) were used to identify the differential proteins; expression analysis of cellular constituents, protein using DAVID Functional Annotation and String online tools for biological pathways, the relationship between the molecular function and tandem mass spectrometry. Results: 2-DE and UBE3A low resistance before and after identified 28 differential protein spots, including 4 protein in low resistance group UBE3A relatively high expression, 24 relatively low expression, remove the identification of the same protein there are 24 different proteins, there are 23 matches with the DAVID DAVID Functional Annotation database; GO class, can enter For biological pathways, cellular component, molecular function annotation classification protein were 23 (100%), 20 (87.7%) and 23 (100%), and in the ubiquitin proteasome system and glucose metabolism classification clear; String analysis showed that these proteins have 23 direct links are directly or indirectly. Conclusion: there is a direct or indirect contact between the 23 proteins, especially in function and glucose metabolism function of the ubiquitin proteasome system changes, which is probably the reason why the change of biological behavior of breast cancer cell lines after UBE3A low resistance.

【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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