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胃癌细胞通过提高有氧糖酵解增强耐药性的机制研究

发布时间:2018-07-05 18:50  文章来源:笔耕文化传播

  本文选题:胃癌 + Enolase ; 参考:《浙江大学》2017年博士论文


【摘要】:背景:在我国,胃癌位列所有肿瘤发病率和死亡率的第二位,严重危害我国国民健康。化疗是肿瘤的重要治疗手段之一。顺铂是胃癌化疗的一线用药,但因能引起多药耐药而制约临床疗效。以往研究发现肿瘤细胞增强瓦伯格效应增强顺铂耐药,但也有报导耐药细胞糖酵解水平降低。故阐明胃癌顺铂耐药细胞糖酵解特征以及与耐药性的关系能为逆转耐药提供治疗思路。目的:研究胃癌顺铂天然耐药和获得性耐药细胞的糖酵解特征,阐明糖酵解对耐药性的影响和作用机制,研究耐药细胞通过何种机制调控糖酵解。方法:前期通过浓度梯度诱导法建立胃癌获得性耐药细胞BGC823/DDP株,选取MGC803细胞株作为天然耐药模型,BGC823细胞株作为药敏感细胞。运用比色法检测敏感细胞和耐药细胞糖酵解相关的底物和产物,包括葡萄糖消耗量、丙酮酸和乳酸产量。通过MTS法、流式凋亡和Western Blot检测敏感细胞和耐药细胞对糖剥夺和糖酵解抑制剂2-DG的敏感性以及两种处理与耐药性的影响。用二维电泳-质谱法得到敏感细胞和耐药细胞的蛋白差异表达谱,并通过蛋白功能聚类分析筛选出与糖酵解相关的差异蛋白。用siRNA敲除该蛋白,比色法检测耐药细胞葡萄糖消耗量、丙酮酸、乳酸和ATP产量。用MTS法、流式凋亡和Western Blot检测敲除差异蛋白后对耐药细胞顺铂敏感性的影响。将敏感细胞过表达差异蛋白,用MTS和Western Blot检测顺铂敏感性。用RT-qPCR、Western Blot及双荧光素酶报告实验等手段研究耐药细胞对差异表达蛋白的调控机制。免疫组化法分析糖酵解相关的差异蛋白在邻近的非肿瘤胃粘膜组织和胃癌组织表达的差异性,及与病人临床特征的关系。结果:天然耐药细胞MGC803和后天获得性耐药细胞BGC823/DDP比敏感细胞BGC823糖酵解水平增强,其葡萄糖消耗量更大,丙酮酸和乳酸的产量更高。耐药细胞对糖剥夺或糖酵解抑制剂2-DG更敏感。抑制糖酵解能逆转耐药细胞的耐药性。二维电泳-质谱分析发现糖酵解过程中的烯醇化酶ENO1(.Enolase 1)在BGC823/DDP细胞中高表达,Western Blot验证结果一致。敲除ENO1后耐药细胞葡萄糖消耗下降,且丙酮酸、乳酸和ATP产量下降,细胞耐药性降低。敏感细胞BGC823过表达ENO1能提升糖酵解水平,抵抗顺铂引起的凋亡。RT-qPCR结果显示耐药细胞中ENO1mRNA水平较敏感细胞轻度下调,说明并非转录水平调节。放线菌酮实验发现敏感细胞和耐药细胞的ENO1蛋白半衰期没有明显差异。提示ENO1可能受转录后调控。应用micRNA靶点预测得到ENO1可能是miR-22的靶点,RT-qPCR检测发现miR-22在耐药细胞中呈低表达。Western Blot检测提示当在耐药细胞过表达miR-22模拟物或在敏感细胞使用miR-22抑制剂,则能下调或上调ENO1的表达。双荧光素酶报告实验提示miR-22可结合ENO1的3'-UTR区,抑制ENO1 mRNA的功能。耐药细胞过表达miR-22模拟物或敏感细胞使用miR-22抑制剂,能相应下调或提升细胞糖酵解水平。免疫组化结果发现,ENO1在胃癌中高表达,在非胃癌组织中低表达,ENO1预示着更差的总生存期。结论:本文发现了胃癌顺铂耐药细胞糖酵解水平上升、更依赖糖酵解,并通过高表达烯醇化酶ENO1上调糖酵解水平。抑制糖酵解途径能够逆转耐药。ENO1在耐药细胞中高表达源于miR-22的转录后调控,故miR-22和ENO1可作为抗多药耐药的治疗靶点。
[Abstract]:Background: in China, gastric cancer ranks second of all tumor incidence and mortality in China, which seriously endangers the national health of our country. Chemotherapy is one of the important treatments for cancer. Cisplatin is the first line of chemotherapy for gastric cancer, but it can cause multidrug resistance to restrict clinical efficacy. It is reported that the glycolysis of cisplatin resistant cells in gastric cancer and the relationship with drug resistance can provide therapeutic ideas for reversing drug resistance. Objective: To study the characteristics of glycolysis in natural and acquired resistant cells of cisplatin, and to clarify the effect and mechanism of glycolysis to drug resistance. To study the mechanism of glycolysis by drug resistant cells. Methods: the BGC823/DDP strain of gastric cancer cells was established by the concentration gradient induction method. The MGC803 cell line was selected as the natural drug resistance model and the BGC823 cell line was used as the drug sensitive cell. The colorimetric assay was used to detect the substrates related to the glycolysis of sensitive cells and drug resistant cells. Products, including glucose consumption, pyruvate and lactic acid production. Sensitivity of sensitive and drug-resistant cells to glucose deprivation and glycolysis inhibitor 2-DG by flow apoptosis and Western Blot by MTS method, and the effects of two treatments and drug resistance on drug resistance. Differential expression of protein in sensitive and drug-resistant cells was obtained by two-dimensional electrophoretic mass spectrometry The differential proteins associated with glycolysis were screened by protein functional clustering analysis. The protein was knocked out by siRNA, and the production of glucose consumption, pyruvic acid, lactic acid and ATP were detected by colorimetric assay. The sensitivity of cisplatin sensitivity to drug-resistant cells was detected by MTS, flow apoptosis and Western Blot to detect the sensitivity of cisplatin in drug resistant cells. Differential proteins were overexpressed and the sensitivity of cisplatin was detected by MTS and Western Blot. The regulatory mechanism of drug-resistant cells to differentially expressed proteins was studied by RT-qPCR, Western Blot and double luciferase reporter assay. Immunohistochemical analysis was used to analyze the differences in the expression of glycolytic related proteins in adjacent non swollen ruminal and gastric tissues Results: natural drug resistant cells MGC803 and acquired acquired resistance cells (BGC823/DDP) are stronger than sensitive cells BGC823 glycolysis, with greater glucose consumption and higher production of pyruvate and lactic acid. Drug resistant cells are more sensitive to sugar deprivation or glycolysis inhibitor 2-DG. Inhibition of glycolysis can be reversed. Drug resistance of drug-resistant cells. Two-dimensional electrophoretic mass spectrometry (two-dimensional electrophoretic mass spectrometry) found that the enolase ENO1 (.Enolase 1) was highly expressed in BGC823/DDP cells, and the results of Western Blot were consistent. After knockout ENO1, the glucose consumption decreased, and pyruvic acid, lactic acid and ATP production decreased, and cell resistance decreased. Sensitive cell BGC823 over table ENO1 can increase glycolysis level and resistance to cisplatin induced apoptosis.RT-qPCR results show that ENO1mRNA levels in drug-resistant cells are slightly lower than those of sensitive cells, indicating that no transcriptional regulation. There is no significant difference in the ENO1 protein half-life of sensitive and drug-resistant cells. It suggests that ENO1 may be regulated after transcriptional regulation. ENO1 may be the target of miR-22 by micRNA target prediction. RT-qPCR detection shows that miR-22 is low expression of.Western Blot in drug-resistant cells, suggesting that when miR-22 mimics are overexpressed in drug-resistant cells or miR-22 inhibitors are used in sensitive cells, ENO1 can be downregulated or up-regulated. The double Luciferase Report experiment suggests miR-22 junction The 3'-UTR region of ENO1, which inhibits the function of ENO1 mRNA. Drug resistant cells overexpress miR-22 mimics or sensitive cells using miR-22 inhibitors, which can correspondingly decrease or elevate the level of glycolysis. The immunohistochemical results show that ENO1 is highly expressed in gastric cancer and low expression in non gastric cancer tissues, and ENO1 indicates a worse total survival time. Conclusion: This article is a conclusion: This article hair hair The glycolysis level of cisplatin resistant cells in gastric cancer is rising, more dependent on glycolysis, and up regulation of glycolysis through high expression enolase ENO1. Inhibition of glycolysis pathway can reverse the post transcriptional regulation of the high expression of drug-resistant.ENO1 in drug-resistant cells, so miR-22 and ENO1 can be used as therapeutic targets for anti multidrug resistance.
【学位授予单位】:浙江大学
【学位级别】:博士
【学位授予年份】:2017
【分类号】:R735.2

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