人肺腺癌吉非替尼耐药细胞株中微小RNA的表达谱

目的:分析人肺腺癌吉非替尼耐药细胞株PC9/AB2与亲本细胞株PC9中微小RNA(microRNA,miRNA)表达谱的差异.方法:应用Agilent human miRNA芯片分别检测PC9/AB2细胞与PC9细胞miRNA的表达谱,随后采用Agilent Feature Extraction软件分析并筛选差异表达的miRNA,应用生物信息软件预测筛选出的差异表达miRNA的潜在靶基因.应用实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)验证芯片检测结果.结果:与PC9细胞比较,PC9/AB2细胞中有36个miRNA表达上调＞2倍,有24个miRNA表达下调＞2倍.RFQ-PCR验证了芯片的结果.软件预测发现,16个miRNA有潜在靶基因,其中11个miRNA的靶基因＞100个.结论:筛选获得的差异表达miRNA可能通过调控其靶基因而参与人肺腺癌细胞对吉非替尼的耐药.     

肺腺癌吉非替尼获得性耐药相关microRNAs的筛选鉴定

背景与目的肺腺癌吉非替尼获得性耐药严重影响了肺癌的治疗效果,microRNA在肺腺癌吉非替尼获得性耐药中的作用及其机制尚不清楚。本研究筛选与肺腺癌获得性吉非替尼耐药相关的microRNAs。方法以吉非替尼敏感肺癌细胞PC9与吉非替尼耐药肺癌细胞PC9/AB11为细胞模型,观察二者的形态学差异,流式细胞仪检测二者的细胞周期,计算它们的倍增时间,MTT法检测吉非替尼对两种细胞的IC50,应用microRNA芯片检测和筛选与吉非替尼耐药相关的microRNAs,并进行real-timePCR验证。结果 PC9细胞与PC9/AB11细胞形态差异明显,在细胞周期、倍增时间和吉非替尼对其的IC50上均具有统计学差异。microRNA芯片结果显示,与PC9相比,耐药肺癌细胞株PC9/AB11中有4个microRNAs表达水平明显上调,有9个microRNAs表达水平明显下调。经real-timePCR验证,microRNA-138在PC9/AB11中表达明显下调,与芯片结果一致。结论 PC9和PC9/AB11细胞株microRNA表达谱存在明显差异,初步筛选到了13个与肺腺癌吉非替尼耐药密切相关的microRNAs,为进一步深入研究microRNA在肺腺癌吉非替尼获得性耐药中的作用及其分子机制提供了实验依据和理论基础。     

非小细胞肺癌吉非替尼耐药相关miRNAs的筛选鉴定

  目的  miRNA是一类通过结合mRNA调节基因表达的非编码单链小分子RNA,本研究目的是探讨非小细胞肺癌（NSCLC）中miRNA与吉非替尼耐药的关系。  方法  CCK8法检测NSCLC吉非替尼耐药细胞PC9/GR相对于亲本细胞PC9的耐药倍数;miRNA芯片检测PC9/GR与PC9中miRNA的表达差异;RT-PCR验证miRNA芯片结果。将差异表达的miRNA模拟物/抑制剂转染至PC9/GR中,观察其对吉非替尼敏感性的影响。  结果  吉非替尼对PC9和PC9/GR的IC₅₀值分别为42.89 nmoL/L和3.87 μ moL/L,耐药倍数为90.23倍。miRNA芯片结果显示,PC9/GR与PC9比较55条有差异表达miRNAs（P < 0.01）,其中在PC9/GR上调的miRNAs有21条,包括miRNA-1 246、miRNA-125b等;下调的miRNAs有34条,包括miRNA-224、miRNA-125a~5p等。RT-PCR进一步验证其中9条miRNAs,有8条与芯片结果趋势一致。将上述8条miRNAs的模拟物/抑制剂转染至PC9/GR中,发现miRNA-125a~5p模拟物可降低吉非替尼敏感性。  结论  PC9/GR与PC9的miRNA表达存在差异,miRNA可能与NSCLC吉非替尼耐药相关,miRNA-125a~5p可促进PC9/GR对吉非替尼产生耐药。      

人非小细胞肺癌细胞EGFR基因启动子甲基化与吉非替尼敏感性之间的相关性研究

目的 探讨EGFR基因启动子甲基化水平与人非小细胞肺癌（NSCLC）细胞株对吉非替尼敏感性之间的相关性。方法 用不同浓度吉非替尼分别作用于NSCLC细胞株HCC827、H1650、H1975、H358、H1299、A549后,CCK-8法检测细胞增殖抑制率,DNA直接测序法、实时荧光定量PCR法、免疫印迹法和甲基化特异性PCR法分别检测上述NSCLC细胞株EGFR基因突变、EGFR mRNA、EGFR蛋白表达和启动子区甲基化状态。结果 CCK-8法检测结果显示,19外显子缺失突变的HCC827细胞对吉非替尼最敏感,而同为19外显子缺失突变的H1650细胞对吉非替尼不敏感;野生型的H358细胞对吉非替尼中度敏感,其敏感性甚至超过19外显子突变的H1650细胞,而同为EGFR野生型的H1299、A549细胞对吉非替尼敏感性较差。吉非替尼处理72h后,HCC827细胞与H358细胞相比、HCC827细胞和H358细胞与其他4株细胞相比,IC50值均有显著性差异（P＜0.05）。HCC827细胞EGFR启动子为未甲基化状态,其EGFR蛋白和mRNA表达最高;H358细胞为部分甲基化,其EGFR蛋白和mRNA为中等表达;其他4个细胞株均为高甲基化状态,EGFR蛋白和mRNA呈低表达;HCC827细胞的EGFR表达水平较H358细胞高,HCC827和H358细胞的EGFR表达较其他4个细胞株高,差异均有统计学意义（P＜0.05）。结论 EGFR基因启动子区高甲基化可能下调EGFR基因的表达水平,从而降低NSCLC对吉非替尼的敏感性;对该基因的甲基化检测可能对预测吉非替尼治疗NSCLC疗效有一定的临床指导意义。     

上皮间质转化在非小细胞肺癌EGFR-TKIs获得性耐药中的作用研究

目的 探讨上皮间质转化（EMT）在非小细胞肺癌（NSCLC）对表皮生长因子受体 酪氨酸激酶抑制剂（EGFR TKIs）获得性耐药中的作用及可能机制。方法 选用EGFR基因19号外显子突变型吉非替尼耐药细胞PC9／AB和EGFR野生型厄洛替尼耐药细胞H460/ER,通过基因转染获得E-cadherin稳定过表达细胞PC9／AB-CDH1和H460ER-CDH1。四甲基偶氮唑盐（MTT）法检测细胞的增殖情况,划痕实验及Transwell侵袭实验检测细胞迁移和侵袭能力,实时荧光定量PCR（qRT-PCR）和蛋白印迹法检测EMT相关分子、EGFR信号通路分子的mRNA和蛋白表达水平。结果 PC9／AB和H460/ER细胞未发生T790M突变和c-Met基因扩增,但发生了EMT,表现为E-cadherin表达降低和Vimentin表达增加。通过基因转染提高E-cadherin表达水平能逆转PC9／AB和H460／ER耐药细胞的EMT,使其对EGFR TKIs的敏感性增加, PC9／AB-CDH1细胞较PC9／AB对吉非替尼的敏感性增加约11.4倍,其半数抑制浓度（IC50）分别为（0.70±0.22） μmol／L和（8.68±0.44）μmol／L,差异有统计学意义（P＜0.05）; H460／ER-CDH1较H460／ER对厄洛替尼的敏感性增加约6.1倍,其IC50分别为（7.51±1.12）μmol／L和（53.72±12.95）μmol／L,差异有统计学意义（P＜0.05）。同时,逆转EMT后,细胞的EGFR、p-EGFR的mRNA和蛋白表达量均增加,差异有统计学意义（P＜0.05）。结论 阻断耐药细胞的EMT可逆转NSCLC对EGFR-TKIs的获得性耐药,EMT在NSCLC对EGFR-TKIs的获得性耐药中起重要作用,其机制可能与EGFR磷酸化水平降低有关。     

肺癌细胞DAPK基因启动子甲基化与吉非替尼敏感性的相关性研究

目的 探讨死亡相关蛋白激酶（DAPK）基因启动子区域甲基化对非小细胞肺癌（NSCLC）细胞株中表皮生长因子酪氨酸激酶抑制剂（EGFR-TKI）敏感性的影响。方法 采用不同浓度吉非替尼作用2株EGFR外显子19缺失突变型（H1650、PC9）和2株EGFR野生型（A549、H520）NSCLC细胞。5-氮杂-2′-脱氧胞苷（5-aza-CdR）去甲基化处理,用CCK-8法检测细胞增殖率,流式细胞技术检测细胞凋亡率,荧光定量PCR法检测DAPK mRNA表达情况,甲基化特异性PCR（MSP）法检测DAPK启动子区甲基化状态。结果 吉非替尼对4种细胞株均存在不同程度的增殖抑制作用,呈浓度依赖性。5-aza-CdR去甲基化后,H1650细胞及H520细胞对吉非替尼的敏感性较前增强（P＜0.05）;流式细胞仪检测显示,H1650、H520细胞凋亡率上升较明显（P＜0.05）。未经5-aza-CdR处理的细胞株中,吉非替尼敏感型的PC9、A549细胞株存在DAPK mRNA高表达,其基因启动子处于非甲基化状态;吉非替尼耐药型的H1650、H520细胞株DAPK mRNA表达水平较低,DAPK启动子处于高甲基化状态。5-aza-CdR去除H1650及H520细胞的DAPK基因启动子区甲基化后,DAPK基因表达及对吉非替尼的敏感性较前显著升高（P＜0.05）;而吉非替尼敏感的PC9及A549细胞株经5-aza-CdR处理后未见明显改变（P＞0.05）。结论 抑癌基因DAPK启动子区域的高甲基化能够下调DAPK基因的表达,从而降低NSCLC对吉非替尼的敏感性。对DAPK基因进行甲基化状态检测,可能为临床上预测吉非替尼疗效提供新的手段。     

β-榄香烯体外逆转人肺腺癌细胞PC9耐吉非替尼的研究

[目的]探讨β-榄香烯体外逆转人肺腺癌细胞PC9耐吉非替尼的作用及其机制.[方法]噻唑蓝(MTT)比色法测定吉非替尼对PC9/ZD的耐药性,计算耐药倍数.MTT法检测无细胞毒的β-榄香烯浓度,并检测此浓度逆转PC9/ZD的作用,计算逆转倍数.Western Blot检测耐药相关蛋白P-gp、survivin的表达情况.[结果]吉非替尼对PC9细胞的IC50为0.21 μmol/L,对PC9/ZD细胞的IC50为5.45μmol/L,耐药倍数为25.9.当β-榄香烯浓度低于10μg/ml时,对PC9及PC9/ZD的抑制率均小于10％,没有细胞毒性.β-榄香烯10μg/ml可逆转PC9/ZD对吉非替尼的耐药性,逆转倍数为2.68,同时细胞内P-gP及survivin的表达水平明显降低.[结论]β-榄香烯具有逆转PC9/ZD细胞耐吉非替尼的作用,可能与下调耐药细胞内P-gp、survivin表达水平相关.     

吉非替尼敏感性不同的非小细胞肺癌中miRNA-7表达的差异及临床意义

目的检测吉非替尼敏感性不同的非小细胞肺癌中miR-7的表达差异,并探讨其临床意义。方法采用吉非替尼(Gefitinib)药物大剂量冲击法诱导H827,建立耐吉非替尼肺癌细胞亚系H827-7/GR,有限稀释法将H827-7/GR细胞单克隆化,CCK-8法检测耐药前后细胞株及各单克隆细胞对吉非替尼的敏感性;RT-PCR方法检测H827、H827-7/GR和耐药单克隆细胞株以及其他对吉非替尼敏感性不同的肺癌细胞株A549、H358、H1299、H1650和H1975中miR-7的表达差异。结果 H827/GR耐药指数大于100;获得的6株耐药单克隆细胞株对吉非替尼的半生长抑制浓度(the half growth inhibition concentration,IC50)值不同;和吉非替尼敏感株H827相比,诱导的耐药细胞株H827/GR、耐药单克隆细胞株和吉非替尼耐药株A549、H358、H1299、H1650和H1975中miR-7的相对表达水平均降低(P<0.05)。结论在非小细胞肺癌中,耐药细胞中miR-7的相对表达水平均较敏感细胞系降低,提示miR-7的低表达可能与非小细胞肺癌耐药性相关,其可能是潜在的肺癌药物敏感性预测分子标志物。     

MircoRNA-214对吉非替尼敏感及耐药细胞增殖和凋亡的影响

摘 要：［目的］ 研究mircoRNA-214（miR-214）对PC9肺腺癌及吉非替尼耐药细胞（PC9/GR）增殖和凋亡的影响。［方法］ 在PC9细胞中转染miR-214模拟物及PC9/GR中转染miR-214抑制剂,使用定量逆转录PCR（qRT-PCR）检测其表达。MTT检测细胞转染miR-214模拟物或其抑制剂后的存活及增殖。在PC9细胞中顺时转染miR-214模拟物以检测上调miR-214对PC9细胞耐药性的影响,在PC9/GR细胞中顺时转染miR-214抑制物以检测下调miR-214对PC9/GR细胞耐药性的影响,并用流式细胞仪检测细胞的凋亡。Western blotting检测PTEN在PC9和PC9/GR细胞中的表达。构建PTEN 3’-UTR荧光素酶报告质粒验证miR-214的靶基因;建立异种移植模型检测miR-214抑制物对肺癌移植瘤的影响。［结果］ PC9细胞中miR-214低表达,上调miR-214的表达后PC9细胞对吉非替尼的敏感性降低,并且抵抗吉非替尼诱导的凋亡;而在PC9/GR细胞中低表达miR-214后,下调miR-214增加PC9/GR细胞对吉非替尼的敏感性,并且可以增强吉非替尼诱导的凋亡作用。荧光素酶报告载体实验证实PTEN是miR-214在细胞内的靶基因。动物异种移植模型表明miR-214抑制物可以增强PC9/GR对吉非替尼的敏感性。［结论］ MiR-214可能通过靶基因PTEN调控吉非替尼的获得性耐药。     

吉非替尼对肺癌细胞株A549和H1975放射敏感度的影响及其机制

目的 本研究旨在探讨小分子表皮生长因子受体（epidermal growth factor receptor, EGFR）酪氨酸激酶抑制剂吉非替尼是否能增加肺癌细胞株A549和H1975的放疗敏感度及其机制。方法 选取两种非小细胞肺癌细胞株A549和H1975,分为单纯X线组和X线+吉非替尼组。单纯X线组采用单纯X线照射,X线+吉非替尼组经10 μmol/L吉非替尼作用24 h后行X线照射。两株细胞不同分组细胞,采用克隆形成实验检测放射敏感度,免疫荧光激光共聚焦显微镜观察X线照射后不同时间点细胞核中磷酸化H2AX（γ-H2AX）亮点在细胞中的定位情况,Western blot法检测放疗后胞质胞核蛋白中EGFR的表达。结果 克隆形成实验中,A549细胞X线+吉非替尼组在各放疗剂量点的SF2值（0.3475）低于单纯X线组（0.4833）;H1975细胞X线+吉非替尼组与单纯X线组在各放疗剂量点的SF2值分别为0.3094和0.3207,无明显差异。免疫荧光结果显示,照射4 Gy后各时间点X线+吉非替尼组A549细胞核中γ-H2AX亮点相比单纯X线多;单纯X线组和X线+吉非替尼组H1975细胞γ-H2AX亮点在各时间点无明显差异; Western blot结果显示,A549细胞经4Gy照射后EGFR有入核现象,而预先经吉非替尼处理再接受4Gy照射,EGFR蛋白绝大部分位于细胞质内;H1975细胞,单纯X线组和X线+吉非替尼组EGFR蛋白均在细胞质中表达,胞核中几乎没有,且两组无明显差异。结论 吉非替尼能增加肺癌细胞株A549的放射敏感度,可能与阻止放疗后EGFR入核进行双链断裂（double strand break,DSB）修复有关;对H1975细胞无影响,与其放疗后EGFR不入核相关。     

Glutamate Promotes Cell Growth by EGFR Signaling on U-87MG Human Glioblastoma Cell Line

Accumulating evidences suggest that glutamate plays a key role in the proliferation and invasion of malignant glioblastoma (GBM) tumors. It has been shown that GBM cells release and exploit glutamate for proliferation and invasion through AMPA glutamate receptors. Additionally, amplification of the epidermal growth factor receptor (EGFR) gene occurs in 40–50% of GBM. Since, PI3K/Akt is considered one of the main intracellular pathways involved in EGFR activation, AKT functions could trigger EGFR signaling. Thus, we investigated whether EGFR-phospho-Akt pathway is involved on the glutamate inducing U-87MG human GBM cell line proliferation. For these purpose, we treated the U-87MG cell line with 5 to 200 mM of glutamate and assessed the number of viable cells by trypan blue dye exclusion test. An increase in cell number (50%) was found at 5 mM glutamate, while the addition of DNQX (500 μM), an antagonist of AMPA receptor, inhibited the effect of glutamate on the U87-MG cells proliferation. Also, at 5 mM glutamate we observed an increase on the EGFR and phospho-Akt contents evaluated by immunohistochemistry. Moreover, U-87MG cells treated with glutamate exhibited an increase about 2 times in the EGFR mRNA expression. While, in the presence of the anti-EGFR gefitinib (50 μM) or the PI3K inhibitor wortmannin (5 μM), the U-87MG proliferation was restored to control levels. Together, our data suggest that glutamate signaling mediated by AMPA receptor induces U-87MG human GBM cell line proliferation via EGFR-phospho-Akt pathway.     

EGFR Antisense Oligonucleotides Encapsulated withNanoparticles Decrease EGFR,MAPK1 and STAT5 Expressionin a Human Colon Cancer Cell Line

Epidermal growth factor receptor (EGFR) is over-expressed in several human cancers. This would suggestthat inhibition of EGFR is a reasonable approach for cancer treatment. In this study we investigated EGFRblocking and its effects on the mediated signaling such as MAPK and STATb in HT29 cells. For this aim weused FITC-labeled EGFR antisense oligonucleotides encapsulated with PAMAM nanoparticles to inhibit EGFRexpression. Cellular uptake of antisense was investigated by fluorescence microscopy and flow cytometry analysis.The effect of EGFR antisense on the expression of EGFR in HT29 cells was examined by real time PCR andWestern blots, which showed that antisense encapsulated with PAMAM decreased the level of EGFR mRNAand protein. In addition, real time PCR results confirmed that EGFR inhibition had an effective role in thereduction of EGFR dependent downstream genes. In conclusion, EGFR antisense encapsulated with PAMAMnanoparticles down regulated EGFR and EGFR-mediated genes.     

Antitumor Efficacy of SLPI Promoter-Controlled Expression of Artificial microRNA Targeting EGFR in a Squamous Cell Carcinoma Cell Line

The purpose of this study was to develop a recombinant adenovirus with secretory leukoprotease inhibitor (SLPI) promoter-controlled expression for gene therapy of squamous cell carcinoma (SCC). An artificial microRNA targeting epidermal growth factor receptor (EGFR) was designed, and used to construct a replication-defective recombinant adenovirus with SLPI promoter-controlled expression. The silencing efficiency of this vector (Ad-SLPI-EGFRamiR) was detected in Hep-2 cells. Western blotting showed that the expression of 170 kD EGFR was significantly reduced in Hep-2 cells 72 h after infection with Ad-SLPI-EGFRamiR. At a multiplicity of infection (MOI) of 200 pfu/cell, proliferation of Hep-2 cells was highly inhibited by Ad-SLPI-EGFRamiR (inhibition rate: ~70%). The apoptosis rate of Hep-2 cells at 72 h after infection with Ad-SLPI-EGFRamiR at a MOI 35 pfu/cell was 32.8%. The adenovirus constructed was able to specifically inhibit the growth of SCC cells in vitro.     

EGFR突变与非小细胞肺癌

表皮生长因子受体(EGFR)为酪氨酸激酶活性的受体,由生长因子激活,调节上皮细胞的生长分化。近年研究发现62%NSCLC中存在EGFR高表达。突变的NSCLC中,50%～80%对Gefitinib、Erlotinib有反应。EGFR突变改变Gefitinib、Erlotinib敏感性,并与NSCLC耐药相关。文章对EGFR与NSCLC的关系作一综述。     

EGFR Inhibitor Enhances Cisplatin Sensitivity of Oral Squamous Cell Carcinoma Cell Lines

Epidermal growth factor receptor (EGFR) is involved in multiple aspects of cancer cell biology. EGFR has already been identified as an important target for cancer therapy, with various kinds of EGFR inhibitors currently used in treatment of several human cancers. Recently, EGFR and its downstream signaling pathways were identified as being associated with cisplatin sensitivity. In addition, EGFR inhibitors have shown significant promise for patients who failed cisplatin-based therapy. In this study, we investigated whether treatment with an EGFR inhibitor improves cisplatin sensitivity in oral squamous cell carcinoma (OSCC) cell lines. The effects of a combination of AG1478, a specific EGFR tyrosine kinase inhibitor, with cisplatin were evaluated in cultured OSCC cell lines and cisplatin-resistant sublines. Higher expression of EGFR and p-EGFR was found in the two cisplatin-resistant cell lines compared with the corresponding parental cell lines. In addition, augmented inhibition of OSCC cell growth by the combination of AG1478 with cisplatin was found in both cell lines. These results suggest that the combination of an EGFR inhibitor and cisplatin may be useful as a rational strategy for the treatment of patients with oral cancer with acquired cisplatin resistance.     

EZB-ICR Cell Line: A New Established and Characterized Oral Squamous Cell Carcinoma Cell Line From Tongue

Background: Tongue cancer is one of the most aggressive forms of oral squamous cell carcinoma which needs more investigations. Herein, we aimed to establish and characterize a tongue cancer cell line. Methods: Tumor tissue was obtained from a 70-year-old woman with tongue cancer. The established cell line named as EZB-ICR and characterized for doubling time, expression of specific markers, HPV corporation and migration status using flow cytometry, immunofluorescence staining, multiplex PCR, and migration assay. Results: EZB-ICR was negative for expression of mesenchymal specific markers, cytokeratin19, pan-cytokeratin, vimentin and EPCAM, but was positive for S100 and Nestin. No appearance of human papilloma virus DNA was seen. The doubling time of EZB-ICR was 31 hours and migration assay confirmed its metastatic nature. Conclusion: To the best of our knowledge, EZB-ICR is the first tongue human cancer cell line established in Iran, and its features make it appropriate for cancer-based in vitro studies and contribute to more studies on tongue cancer.     

Treatment Options for EGFR T790M-Negative EGFR Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer

The introduction of first- and second-generation EGFR-tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib and afatinib) for the treatment of advanced EGFR-mutant non-small cell lung cancer (NSCLC) has dramatically improved patients’ prognosis and quality of life (QoL). Unfortunately, after an initial and sometimes durable benefit from EGFR-TKI therapy, all patients with EGFR-mutant lung cancer eventually become resistant to the treatment and experience disease progression. In approximately 50% of these patients, genomic alterations in the EGFR kinase domain resulting in the mutant T790M are responsible for the resistance and this has led to the development of novel EGFR inhibitors active against mutant-T790M EGFR. The remaining 50% of patients with acquired resistance (AR) to EGFR-TKIs do not harbour the T790M mutation. In these cases, other mechanisms are involved in the development of AR such as perturbations of downstream pathways (e.g. K-RAS mutations), activation of alternative bypassing pathways (including c-Met, AXL, PIK3CA, BRAF), or histologic transformation. This review summarizes the main treatment strategies for this particular and heterogeneous group of “T790M-negative” patients.

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