Filamin A影响EGFR T790M突变肺腺癌H1975细胞对三代EGFR-TKIs敏感性的研究

发布时间：2018-01-12 10:01

  本文关键词：Filamin A影响EGFR T790M突变肺腺癌H1975细胞对三代EGFR-TKIs敏感性的研究　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 非小细胞肺癌 细丝蛋白A 表皮生长因子受体 酪氨酸磷酸化 AZD9291

【摘要】：目的:肺癌是我国肿瘤相关死亡的主要病因,在男性恶性肿瘤中其发病率及死亡率最高,女性发病率居第二位,但其死亡率仍为女性肿瘤死亡的首位。非小细胞肺癌(NSCLC)在肺癌中最常见,约占85%。针对其治疗方法主要有手术、化疗、放疗、分子靶向治疗以及免疫治疗等。近年来化疗及手术治疗的水平虽有所提高,但其5年生存率仅达15%。分子靶向治疗开启了肺癌治疗的新纪元,即以肿瘤原癌基因或其信号通路分子为治疗靶点,有针对性对肿瘤细胞发挥抑制作用,降低对正常细胞的损伤,为肿瘤患者带来了福音。表皮生长因子受体(EGFR)是一类具有酪氨酸激酶活性的受体,属于Erb B受体家族的一员。EGFR胞外区与配体如表皮生长因子(EGF)结合形成同源二聚体,在酪氨酸残基上发生磷酸化,进一步活化下游信号通路,如MAPK/ERK、PI3K/AKT、JAK/STAT等,参与调控肿瘤细胞增殖、侵袭、转移及血管生成等。研究证实,EGFR活化突变与NSCLC的进展和预后不良相关。最常见的EGFR激活突变以19外显子缺失或21外显子L858R替代突变的形式发生,在西方人群中大约10%-15%非小细胞肺癌的患者携带着EGFR激活突变,而在亚洲人群约占40%。针对其分子治疗的靶向药物已成功应用于临床,第一代EGFR酪氨酸激酶抑制剂(EGFR-TKIs),如吉非替尼和厄洛替尼广泛用于晚期NSCLC患者的一线治疗方案,对比以铂类为主的双药化疗能够显著延长中位无疾病进展生存期(PFS),改善生活质量。然而,大部分患者在应用9-14个月治疗后,往往将会面临EGFR-TKIs耐药。最常见的获得性耐药机制是T790M突变,增加了与ATP的亲和力从而降低抑制剂疗效。此外,还包括MET基因扩增、小细胞肺癌转化、EGFR突变基因丢失等。另外,约30%患者对EGFR-TKIs表现为原发性耐药。针对这一耐药问题,后续又研发出了第二代阿法替尼及第三代奥希替尼(AZD9291),其中AZD9291是一个新的口服药,有效的、选择性的抑制EGFR敏感突变和T790M耐药突变的不可逆抑制剂,对野生型EGFR效果差些。所以,我们进一步来探讨NSCLC患者对EGFR-TKIs的敏感性的问题,为后续治疗提供一定的指导作用。细丝蛋白A(Filamin A,FLNa)是一种非肌性肌动蛋白结合蛋白,主要存在细胞浆中。FLNa包括一个位于氨基末端的肌动蛋白结合结构域和一个由24个串联重复序列组成的棒状结构域,多层β折叠的结构在这个棒状结构域为蛋白质之间相互作用提供了界面。FLNa通过调节信号传导分子可以改变致瘤微环境。目前,Zhu等研究发现减少FLNa表达量后能够促进肺癌PC-9细胞的增殖、迁移和侵袭;减少FLNa表达量也能导致肺癌PC-9细胞对第一代EGFR-TKIs敏感性下降。然而,患者经过一段时间治疗后往往出现耐药问题,针对其最常见的机制T790M突变,研发出了第三代抑制剂AZD9291,其单苯胺基嘧啶化合物在结构和药理学上不同与其他几代TKIs。为此我们了解一下药物结构不同的TKI与FLNa的相互作用,及细胞对药物敏感性的影响。本研究拟用体外培养EGFR第21外显子敏感突变和20外显子T790M耐药突变的NSCLC H1975细胞株,通过:采用质粒转染技术,构建沉默FLNa的H1975细胞株;应用MTS法、平板克隆、划痕修复试验及侵袭试验检测稳定转染细胞株增殖、迁移和侵袭生物学行为的能力;应用Western blot印迹检测AZD9291对稳定转染细胞信号分子水平的影响,从而探讨FLNa影响EGFR T790M突变肺腺癌H1975细胞对三代EGFR-TKIs的敏感性。方法:1 Western bolt检测稳定转染细胞株中FLNa沉默效果分别将本实验室保存的肺腺癌H1975、H1975/FLNa(KD)及H1975/ctrl细胞接种于35mm培养皿,完全1640培养基培养至贴壁长满。收集细胞并提取蛋白,应用Western blot检测FLNa的水平,以?-actin作为内参蛋白。2 MTS比色分析法检测两组细胞增殖能力分别将H1975/FLNa(KD)及H1975/ctrl细胞接种于96孔板,培养24h,弃去上清液,换用含有不同浓度AZD9291(0、2、4、8、16μmol/L)的完全1640培养基继续培养48h,弃去上清液加入MTS,测定各孔的吸光度(OD)值,并计算细胞生长抑制率和AZD9291的半数抑制浓度(IC50)。3平板克隆法检测两组细胞贴壁后克隆形成变化分别将H1975/FLNa(KD)及H1975/ctrl细胞接种于35mm培养皿,在有无(1μmol/L)AZD9291作用下培养2周,苏木素染色,倒置显微镜下观察并计数细胞克隆形成数量。4划痕修复实验检测两组细胞迁移能力分别将H1975/FLNa(KD)及H1975/ctrl细胞接种于24孔板,培养24h,使用无菌移液器吸头在单层细胞上均匀划痕,两种细胞分别用含有或不含有1μmol/L AZD9291的完全1640培养基继续培养,观察到各组细胞迁移变化无明显差异,故增加药物浓度至2μmol/L,定时观察并拍照记录至划痕愈合。测量宽度变化并计算细胞的迁移率。5侵袭试验检测两组细胞侵袭能力分别将H1975/FLNa(KD)及H1975/ctrl细胞接种于铺有基质胶的Transwell小室的上室,两种细胞分别于含有或不含有1μmol/L AZD9291的无胎牛血清1640培养基中培养,下室放入含10%胎牛血清的1640培养基,继续培养24h后,固定,染色,光镜拍照并计算穿膜细胞数。6 Western blot检测两组细胞信号通路分子活性变化分别将H1975/FLNa(KD)及H1975/ctrl细胞接种于35mm培养皿,完全1640培养基培养至贴壁长满。予以1μmol/L AZD9291进行干预,分别于0h、2h、4h、8h后收细胞,提取总蛋白,Western blot检测p-EGFR、EGFR、p-ERK、ERK、FLNa的蛋白水平,?-actin作为内参蛋白。结果:1稳定转染H1975细胞中FLNa蛋白表达水平的变化Western blot检测两组细胞FLNa蛋白水平,扫描蛋白条带并统计分析,结果:H1975/FLNa(KD)细胞中FLNa的表达量(0.34±0.01)明显低于H1975/ctrl细胞(0.91±0.02),差异具有统计学意义(P0.01)。2稳定转染H1975细胞AZD9291的IC50MTS法检测两组细胞的增殖能力,结果:不同浓度的AZD9291作用下,其对H1975/FLNa(KD)细胞的生长抑制率均高于相应浓度的H1975/ctrl细胞,差异有统计学意义(P0.05);H1975/FLNa(KD)组AZD9291的IC50值(2.17±0.19)明显低于H1975/ctrl组细胞(6.09±0.11),差异具有统计学意义(P0.01)。H1975/FLNa(KD)组对AZD9291的敏感性高于H1975/ctrl组。3稳定转染H1975细胞贴壁克隆生长能力的比较平板克隆法检测两组细胞克隆生长能力,结果:在无AZD9291作用2周后,H1975/FLNa(KD)组细胞克隆形成率(91.18±3.27%)明显低于H1975/ctrl组(143.26±6.49%),差异均有统计学意义(P0.01);在含1μmol/L AZD9291作用2周后,H1975/FLNa(KD)组细胞克隆形成率(7.95±2.02%)明显低于H1975/ctrl组(21.07±3.21%),差异均有统计学意义(P0.01)。4稳定转染H1975细胞迁移能力的比较划痕修复实验检测两组转染细胞迁移能力,结果:在无AZD9291作用下,H1975/FLNa(KD)组细胞在6h、12h、18h的迁移率(5.21±0.85%,12.30±0.32%,19.09±0.66%)均明显低于H1975/ctrl组(13.79±1.88%,33.49±1.92%,50.00±0.00%),差异均有统计学意义(P0.01);在2μmol/L AZD9291作用下,H1975/FLNa(KD)组细胞6h、12h、18h的迁移率(4.07±0.66%,9.26±1.48%,13.07±1.35%)明显低于H1975/ctrl组细胞(7.93±1.05%,21.90±1.50%,33.90±1.27%),差异均有统计学意义(P0.01)。5稳定转染H1975细胞侵袭能力的比较Transwell法检测两组转染细胞侵袭能力,结果:在无AZD9291作用下,H1975/FLNa(KD)组穿膜的细胞数量(127.80±4.49)少于H1975/ctrl组(221.60±5.86),在1μmol/L AZD9291作用下,H1975/FLNa(KD)组穿膜的细胞数量(29.00±2.24)少于H1975/ctrl组(70.20±2.39),差异均有统计学意义(P0.01)。6稳定转染H1975细胞EGFR活化水平及其下游信号分子活性的比较Western blot检测1μmol/L AZD9291作用于两组细胞0h、2h、4h、8h后各蛋白的表达水平,结果:H1975/FLNa(KD)组p-EGFR的水平(0.30±0.02;0.08±0.02;0.10±0.02;0.01±0.00)均明显低于H1975/ctrl组(0.36±0.02;0.44±0.01;0.16±0.01;0.06±0.01),差异具有统计学意义(P0.05);H1975/FLNa(KD)组p-ERK的水平(0.90±0.04;0.31±0.00;0.28±0.01;0.18±0.06)均低于H1975/ctrl组(1.11±0.09;1.42±0.02;1.12±0.12;1.01±0.11),差异具有统计学意义(P0.05)。结论:1下调FLNa表达可加强AZD9291对T790M突变肺腺癌H1975细胞增殖、迁移和侵袭的抑制作用。2下调FLNa通过降低EGFR磷酸化水平,抑制MAPK/ERK信号通路活化,提高H1975细胞对三代EGFR-TKI AZD9291的敏感性。
[Abstract]:Objective: lung cancer is a major cause of cancer death in China, its incidence and mortality is highest in male malignant tumors, the incidence of women ranked second, but the mortality is still female cancer death first. Non small cell lung cancer (NSCLC) is the most common in lung cancer, accounting for about 85%. for the main treatment method there are surgery, radiotherapy, chemotherapy, molecular targeted therapy and immunotherapy. In recent years, chemotherapy and surgical treatment level has improved, but the 5 year survival rate is only 15%. molecular targeted therapy to open a new era in the treatment of lung cancer with tumor oncogene or its signal pathway as a therapeutic target targeted, exert inhibitory effects on tumor cells, reduce the damage of normal cells, to bring the gospel. For patients with tumors of the epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor, B receptor belongs to the Erb family a Member of the extracellular domain of.EGFR and ligands such as epidermal growth factor (EGF) combined with two homodimers, phosphorylated at tyrosine residues, further activation of downstream signaling pathways, such as MAPK/ERK, PI3K/AKT, JAK/STAT etc., involved in the regulation of tumor cell proliferation, invasion, metastasis and blood of Guan Shengcheng et al. Study confirmed that the EGFR mutation and activation the progress and prognosis of NSCLC. The most common adverse related activation of EGFR mutations in exon 19 and exon 21 L858R substitution mutation occurs in the form of the population in the west, approximately 10%-15% of patients with non-small cell lung cancer with EGFR activating mutations, and in the Asian population accounts for about 40%. in molecular therapy target the drug has been successfully used in clinic, the first generation of EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib are widely used for first-line treatment of patients with advanced NSCLC, double chemotherapy compared with platinum based Could significantly prolong the median progression free survival (PFS), to improve the quality of life. However, most of the patients in 9-14 months after treatment, often will face EGFR-TKIs resistance. The most common mechanisms of acquired drug resistance is T790M mutation, increased the affinity to ATP so as to reduce the effect of inhibitors. In addition, also includes MET gene amplification, small cell lung cancer, mutations in the EGFR gene loss. In addition, about 30% of patients with EGFR-TKIs showed primary resistance. The resistance, follow-up and developed the second generation and the three generation of afatinib OHI imatinib (AZD9291), where AZD9291 is a new oral drug, effective, selective inhibition of EGFR sensitive mutant irreversible inhibitor of T790M mutation and resistance, the effect of wild type EGFR. Therefore, we further discuss the sensitivity of NSCLC patients to EGFR-TKIs problems, provide for the subsequent treatment A guiding role of filamin A (Filamin A FLNa) is a nonmuscle actin binding protein, mainly exist in the cytoplasm of.FLNa cells including one located at the amino terminus of actin binding domain and one consisting of 24 tandem repeats of the rod domain structure of multilayer beta sheet in this bar domains for protein-protein interactions provides.FLNa interface through the regulation of signal transduction molecules can change the tumorigenic microenvironment. At present, Zhu found that the reduced FLNa expression could promote lung cancer PC-9 cell proliferation, migration and invasion; reduce the expression of FLNa can also lead to lung cancer PC-9 cells decreased to the first generation of EGFR-TKIs sensitivity. However, after a period of time after treatment, patients often appear problems of drug resistance, the most common mechanism of T790M mutation, developed the third generation of AZD9291 inhibitors, the single anilino pyrimidine. Complexes differ in structure and pharmacological and other generations of TKIs. so we know about the interaction between TKI and FLNa drug structure, and the influence on the drug sensitivity of cells. In this study we used in vitro culture EGFR twenty-first exon and exon 20 NSCLC mutation sensitive cell line H1975, mutants resistant to T790M by using plasmid transfection, H1975 cell line construction of FLNa silencing; using MTS method, plate clone, scratch test and repair the invasion test stable transfected cell line proliferation, migration and invasion ability of behavioral biology; effect of AZD9291 was detected by Western blot blotting of stably transfected cell signal molecular level, so as to explore the effect of FLNa EGFR T790M the mutation sensitivity of lung adenocarcinoma H1975 cells of the three generation of EGFR-TKIs. Methods: 1 FLNa Western bolt were detected in lo2-peg10 cells silence effects respectively preserved in our laboratory Lung adenocarcinoma H1975, H1975/FLNa (KD) and H1975/ctrl cells were inoculated in 35mm culture dish, 1640 complete medium to full adherence. Cells were collected and protein extraction, application of Western blot detection of the level of FLNa, with -actin as the reference protein?.2 MTS assay detected two groups of cell proliferation were H1975/FLNa (KD) and H1975/ctrl cells were seeded in 96 well plates, cultured 24h, the supernatant was discarded and replaced by containing different concentration of AZD9291 (0,2,4,8,16 mol/L) complete 1640 culture medium to culture 48h, the supernatant was discarded into MTS, absorption spectrophotometric determination of each hole (OD) value, and calculate the half inhibition rate of cell growth and AZD9291 inhibitory concentration (IC50) of two groups of.3 cells was determined by plate cloning method after adherent colony formation were H1975/FLNa (KD) and H1975/ctrl cells were inoculated in 35mm culture dish, with and without AZD9291 (1 mol/L) under the action of training 2 weeks, hematoxylin staining, inverted Under a microscope to observe and count the number of.4 cell colony formation migration of wound healing assay was two groups of cells were H1975/FLNa (KD) and H1975/ctrl cells were seeded in 24 well plates, cultured 24h, using a sterile pipette tip evenly in the monolayer of scratches on the two kinds of cells were used, containing or not containing 1 mol/L AZD9291 complete 1640 culture medium to culture, observed cell migration changes had no significant difference, so the increase of drug concentration to 2 mol/L, the timing of wound healing was observed and photographed. To measure the width change and calculate the rate of cell migration.5 invasion capability test two groups of cells were H1975/FLNa (KD) and H1975/ctrl cells with a Transwell cell seeded on Matrigel in the upper chamber, two kinds of cells were in containing or not containing 1 mol/L AZD9291 no 1640 fetal bovine serum culture medium containing 10% fetal ventricular into 1640 bovine serum culture medium, cultured 24h after fixation, staining, light microscope photographs and calculate the change of the two groups of cell signal pathway transmembrane cell number.6 Western blot H1975/FLNa (KD) were detected and H1975/ctrl cells were inoculated in 35mm culture dish, totally 1640 cultured adherent to be covered. 1 mol/L AZD9291 intervention, respectively in 0h, 2h, 4h, 8h after the resumption of cell total protein extraction, Western detection of blot p-EGFR EGFR, p-ERK, ERK, and protein level of FLNa? -actin as the reference protein. Results: FLNa protein expression changes of Western blot levels in two groups were detected the protein level of FLNa 1 cells stable transfection of H1975 cells, scanning protein bands and statistical analysis, results: H1975/FLNa (KD) expression in FLNa cells (0.34 + 0.01) was significantly lower than that of H1975/ctrl cells (0.91 + 0.02), the differences were statistically significant (P0.01).2 AZD9291 I stably transfected into H1975 cells The proliferation ability, C50MTS assay results: two groups of cells at different AZD9291 concentrations of H1975/FLNa (KD) cell growth inhibition rate was higher than the corresponding concentration of H1975/ctrl cells, the difference was statistically significant (P0.05); H1975/FLNa (KD) AZD9291 IC50 value (2.17 + 0.19) was significantly lower than that of H1975/ctrl group cells (6.09 + 0.11), the difference was statistically significant (P0.01).H1975/FLNa (KD) detection plate cloning method sensitivity group AZD9291 was higher than that of H1975/ctrl group.3 transfected H1975 cells adherent growth of clonal growth ability of two groups of cell clones, results in no AZD9291 effect after 2 weeks, H1975/FLNa (KD) cell clone formation rate (91.18 + 3.27%) was significantly lower than that of H1975/ctrl group (143.26 + 6.49%), the differences were statistically significant (P0.01); in containing 1 mol/L AZD9291 for 2 weeks, H1975/ FLNa group (KD) cell clone formation rate (7.95 + 2.02%) significantly Lower than that of group H1975/ctrl (21.07 + 3.21%), the differences were statistically significant (P0.01) compare the wound healing assay was stably transfected.4 H1975 cells migration of transfected cells in two groups of migration, results: in the absence of AZD9291, H1975/FLNa (KD) cells in 6h, 12h, 18h (5.21 + 0.85%, the rate of migration 12.30 + 0.32%, 19.09 + 0.66%) was significantly lower than that of group H1975/ctrl (13.79 + 1.88%, 33.49 + 1.92%, 50 + 0%), the differences were statistically significant (P0.01); in 2 mol/L AZD9291, H1975/FLNa (KD) 6h 12h, 18h group of cells, the migration rate (4.07 + 9.26 + 0.66%. 1.48%, 13.07 + 1.35%) was significantly lower than that of cells in H1975/ctrl group (7.93 + 1.05%, 21.90 + 1.50%, 33.90 + 1.27%), the differences were statistically significant (P0.01).5 stable transfected H1975 cell invasion detection Transwell method compared two groups of transfected cell invasion. Results: in the presence of AZD9291 H1975/FLNa (KD) group 绌胯啘鐨勭粏鑳炴暟閲，

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