Notch信号通路体外介导肺癌细胞表皮生长因子受体酪氨酸激酶抑制剂获得性耐药的分子机制研究  

Activation of Notch-1 enhances epithelial-mesenchymal transition in gefitinib-acquired resistant lung cancer cells

作　　者：谢勉[1] 何朝生[2] 魏慎海[3] 

机构地区：[1]广州医科大学附属第一医院呼吸疾病国家重点实验室,510120 [2]广东省人民医院广东医学科学院肾内科 [3]清华大学附属第一医院胸外科

出　　处：《肿瘤研究与临床》2015年第5期298-304,共7页Cancer Research and Clinic

基　　金：国家自然科学基金（81301999）;广东省自然科学基金（S2013040014688）

摘　　要：目的 研究Notch信号通路在肺癌表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）吉非替尼获得性耐药中的作用机制.方法 选用EGFR 19号外显子缺失突变（DelE746-A750）的人肺癌PC9细胞株以及吉非替尼获得性耐药PC9/AB2细胞株.构建靶向Notch-1的siRNA真核表达载体,转染PC9/AB2细胞.DNA直接测序法检测EGFR基因20号外显子,荧光原位杂交试验（FISH）检测Met基因扩增.Western blot实验检测EGFR、Akt和Erk蛋白、Notch受体和配体、转化生长因子β（TGF-β）受体以及上皮间质标志物E-Cadherin、Vimentin和Snail表达水平.CCK-8法测定吉非替尼对PC9和PC9/AB2细胞的增殖抑制作用.结果 DNA直接测序法和FISH检测排除吉非替尼耐药株PC9/AB2存在T790M突变和MET扩增.Western blot检测显示PC9/AB2细胞中Notch-1表达较原代PC9细胞明显增强;其余Notch受体、配体和TGF-β受体无差异.PC9/AB2细胞上皮细胞性标志物E-Cadherin表达较PC9细胞明显下调,间质细胞性标志物Vimentin和Snail的表达较PC9明显上调.Notch-1 RNA干扰后PC9/AB2细胞Notch-1减弱,E-Cadherin表达上调,Vimentin和Snail表达明显下调.Notch-1RNA干扰后PC9/AB2细胞由细长梭形重新转变为类上皮细胞的鹅卵石形.RNA干扰下调Notch-1表达可部分逆转PC9/AB2细胞对吉非替尼耐药性、减弱锚定-不依赖性生长能力.结论 Notch-1活化在吉非替尼获得性耐药过程中起一定作用,抑制Notch-1表达可部分逆转吉非替尼获得性耐药.Objective To investigate whether the Notch-1 signaling pathway is involved in the acquisition of the epithelial-mesenchymal transition （EMT） phenotype of gefitinib-acquired resistant lung cancer cells.Methods The PC9 cell line （harboring EGFR exon 19 deletion） and PC9/AB2 cells （gefitinibacquired resistant PC9 cells） were used.siRNA targeting Notch-1 eukaryotic expression vector （siNotch-1） was constructed and PC9/AB2 cells were transfected with siNotch-1.The protein expression of EGFR,Akt,Erk,Notch receptors and ligands,TGF-β receptors,E-cadherin,Vimentin,and Snail were detected by Western blot assay.DNA sequencing and fluorescence in situ hybridization （FISH） analysis were processed to detect mutation of EGFR exon 20 and MET amplification,respectively.For cytotoxicity assay,cell viability was assessed by Cell Counting Kit 8.Results Gefitinib resistant cell line PC9/AB2 had no evidence of MET amplification or EGFR T790M mutation.The expression of Notch-1 was upregulated in gefitinib resistant PC9/AB2 cells compared with that in gefitinib-sensitive PC9 cells.There were no significant protein expression differences of other Notch receptors,Notch ligands or TGF-β receptors between both paired cell lines.Western blot results showed that protein expression of E-cadherin was greatly reduced in PC9/AB2 cells,while elevated levels of Vimentin and Snail were observed.A significant reduction of the expression of Snail and Vimentin in Notch-1 siRNA transfected PC9/AB2 cells with increased E-cadherin expression was found by Western blot assay.PC9/AB2 cells displayed a round like cell morphology after Notch-1 siRNA transfection.Silence of Notch-1 decreased colony-formation ability but enhanced sensitivity of gefitinib on PC9/AB2 cells.Conclusion Notch-1 might play a novel role in acquired resistance to gefitinib,which could be reversed by inhibiting Notch-l.

关 键 词：NOTCH 表皮生长因子受体 耐药 上皮-间质转型 

分 类 号：R734.2[医药卫生—肿瘤]