FBW7参与的非小细胞肺癌EGFR-TKIs获得性耐药实验研究

发布时间：2019-03-29 20:55

【摘要】：目的:FBW7(F-box and WD repeat domain-containing7,FBW7)是F-box蛋白家族成员,为SCF型泛素连接酶复合物中识别底物的蛋白分子,介导细胞内多种蛋白质的泛素化降解。FBW7是一个抑癌蛋白,通过调控细胞增殖、分化、周期和凋亡相关蛋白的降解参与了肿瘤发生和发展的众多生物学过程。我们前期研究发现FBW7在Gefitinib敏感的亲本细胞和相应的耐药细胞中存在差异性表达,细胞耐药后FBW7表达减少。我们将通过以下方案研究FBW7调控Gefitinib敏感性的分子机制,并探讨以FBW7为靶点逆转Gefitinib耐药的研究策略。方法:1、Gefitinib从10 nM开始处理PC-9亲本细胞,每2周加倍Gefitinib浓度,连续培养6个月获得耐药细胞PC-9GR,EGFR基因测序和FISH检测PC-9GR是否出现T790M和Met扩增;2、通过PCR和Western blot方法比较亲本细胞与耐药细胞FBW7在mRNA和蛋白质水平的表达情况;3、通过shRNA下调亲本细胞PC-9和HCC827 FBW7表达,观察细胞是否出现Gefitinib耐药性;4、明确FBW7调控Gefitinib敏感性的分子机制,探究Bcl-2家族抗凋亡蛋白Mcl-1在该过程中的作用。结果:1、MTT实验证实PC-9GR对Gefitinib耐药,基因测序和FISH未发现T790M与Met基因扩增;2、亲本细胞FBW7在mRNA和蛋白质两个表达水平均高于相应耐药细胞;3、shRNA慢病毒感染亲本细胞,PCR和Western blot验证FBW7下调效率,MTT、流式细胞术和Western blot等功能学实验证实亲本细胞在FBW7表达下调后对Gefitinib敏感性降低,细胞出现耐药表型;4、亲本细胞和耐药细胞Bcl-2家族蛋白存在差异性表达,其中Mcl-1的差异最显著。FBW7通过降解抗凋亡蛋白Mcl-1和调控Mcl-1半衰期t1/2影响细胞对Gefitinib敏感性。结论:1、FBW7在PC-9和HCC827亲本细胞表达量高于耐药细胞PC-9GR和HCC827GR5、HCC827GR6,并且PC-9GR不存在已知的耐药机制T790M与Met扩增,提示FBW7表达下调可能是Gefitinib耐药的一个新机制;2、下调亲本细胞FBW7表达,PC-9和HCC827对Gefitinib敏感性降低,Gefitinib IC50增加且细胞凋亡比例降低,推测下调FBW7表达可能稳定抗凋亡分子,抗凋亡分子表达在凋亡刺激下不发生减少,最终导致亲本细胞对Gefitinib耐药;3、下调FBW7表达可以稳定抗凋亡蛋白Mcl-1,减少Mcl-1降解,延长Mcl-1的半衰期t1/2,后者可能是导致Gefitinib耐药的关键分子。
[Abstract]:Objective: FBW7 (F-box and WD repeat domain-containing7,FBW7) is a member of the F-box protein family, which is a substrate-recognizing protein molecule in the SF-type ubiquitin ligase complex and mediates the ubiquitin degradation of a variety of proteins in cells. FBW7 is a cancer suppressor protein. The degradation of cell proliferation, differentiation, cycle and apoptosis-related proteins is involved in many biological processes of tumorigenesis and development by regulating cell proliferation, differentiation, cycle and apoptosis-related protein degradation. Our previous studies showed that FBW7 was expressed differently in Gefitinib-sensitive parent cells and corresponding drug-resistant cells, and the expression of FBW7 decreased after drug-resistance. We will investigate the molecular mechanism of FBW7 regulating the sensitivity of Gefitinib and explore the strategies for reversing Gefitinib resistance by targeting FBW7. Methods: 1. PC-9 parent cells were treated with Gefitinib from 10 nM and the concentration of Gefitinib was doubled every 2 weeks. After 6 months of continuous culture, PC-9GR,EGFR gene sequencing and FISH were used to detect the presence of T790M and Met amplification in PC-9GR cells. (2) the expression of FBW7 in parental cells and drug-resistant cells at mRNA and protein levels were compared by PCR and Western blot methods, and the expression of PC-9 and HCC827 FBW7 in parental cells was down-regulated by shRNA to observe the presence of Gefitinib resistance. 4. To clarify the molecular mechanism of FBW7 regulating Gefitinib sensitivity and to explore the role of Bcl-2 family anti-apoptotic protein Mcl-1 in this process. Results: (1) T790M and Met genes were not detected by PC-9GR assay, and T790M and Met genes were not detected by FISH. (2) the expression level of FBW7 in parental cells was higher than that in the corresponding drug-resistant cells in both mRNA and protein levels. PCR and Western blot confirmed the down-regulation efficiency of FBW7. MTT, flow cytometry and Western blot showed that the sensitivity of parental cells to Gefitinib was decreased after down-regulation of FBW7 expression, and the drug-resistant phenotype of the cells was observed. 4. The differential expression of Bcl-2 family proteins existed between parental cells and drug-resistant cells, among which the difference of Mcl-1 was the most significant. FBW7 affected the sensitivity of cells to Gefitinib by degrading anti-apoptotic protein Mcl-1 and regulating the half-life of Mcl-1. Conclusion: 1 the expression level of FBW7 in PC-9 and HCC827 parent cells is higher than that in PC-9GR and HCC827GR5,HCC827GR6, cells, and there is no known mechanism of T790M and Met amplification in PC-9GR, suggesting that down-regulation of FBW7 expression may be a new mechanism of Gefitinib drug resistance. 2. Down-regulated the expression of FBW7, decreased the sensitivity of PC-9 and HCC827 to Gefitinib, increased the number of Gefitinib IC50 and decreased the percentage of cell apoptosis, speculated that the down-regulation of FBW7 expression might stabilize the anti-apoptotic molecules, but the expression of anti-apoptotic molecules did not decrease under the stimulation of apoptosis. In the end, parent cells were resistant to Gefitinib. 3. Down-regulation of FBW7 expression could stabilize anti-apoptosis protein Mcl-1, and decrease Mcl-1 degradation, and prolong the half-life of Mcl-1, which may be the key molecule leading to Gefitinib resistance.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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