肿瘤研究与临床
腫瘤研究與臨床
종류연구여림상
CANCER RESEARCH AND CLINIC
2015年
5期
298-304
,共7页
Notch%表皮生长因子受体%耐药%上皮-间质转型
Notch%錶皮生長因子受體%耐藥%上皮-間質轉型
Notch%표피생장인자수체%내약%상피-간질전형
Notch%Epidermal growth factor receptor%Drug resistance%Epithelial-mesenchymal transition
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.
目的 研究Notch信號通路在肺癌錶皮生長因子受體酪氨痠激酶抑製劑(EGFR-TKI)吉非替尼穫得性耐藥中的作用機製.方法 選用EGFR 19號外顯子缺失突變(DelE746-A750)的人肺癌PC9細胞株以及吉非替尼穫得性耐藥PC9/AB2細胞株.構建靶嚮Notch-1的siRNA真覈錶達載體,轉染PC9/AB2細胞.DNA直接測序法檢測EGFR基因20號外顯子,熒光原位雜交試驗(FISH)檢測Met基因擴增.Western blot實驗檢測EGFR、Akt和Erk蛋白、Notch受體和配體、轉化生長因子β(TGF-β)受體以及上皮間質標誌物E-Cadherin、Vimentin和Snail錶達水平.CCK-8法測定吉非替尼對PC9和PC9/AB2細胞的增殖抑製作用.結果 DNA直接測序法和FISH檢測排除吉非替尼耐藥株PC9/AB2存在T790M突變和MET擴增.Western blot檢測顯示PC9/AB2細胞中Notch-1錶達較原代PC9細胞明顯增彊;其餘Notch受體、配體和TGF-β受體無差異.PC9/AB2細胞上皮細胞性標誌物E-Cadherin錶達較PC9細胞明顯下調,間質細胞性標誌物Vimentin和Snail的錶達較PC9明顯上調.Notch-1 RNA榦擾後PC9/AB2細胞Notch-1減弱,E-Cadherin錶達上調,Vimentin和Snail錶達明顯下調.Notch-1RNA榦擾後PC9/AB2細胞由細長梭形重新轉變為類上皮細胞的鵝卵石形.RNA榦擾下調Notch-1錶達可部分逆轉PC9/AB2細胞對吉非替尼耐藥性、減弱錨定-不依賴性生長能力.結論 Notch-1活化在吉非替尼穫得性耐藥過程中起一定作用,抑製Notch-1錶達可部分逆轉吉非替尼穫得性耐藥.
목적 연구Notch신호통로재폐암표피생장인자수체락안산격매억제제(EGFR-TKI)길비체니획득성내약중적작용궤제.방법 선용EGFR 19호외현자결실돌변(DelE746-A750)적인폐암PC9세포주이급길비체니획득성내약PC9/AB2세포주.구건파향Notch-1적siRNA진핵표체재체,전염PC9/AB2세포.DNA직접측서법검측EGFR기인20호외현자,형광원위잡교시험(FISH)검측Met기인확증.Western blot실험검측EGFR、Akt화Erk단백、Notch수체화배체、전화생장인자β(TGF-β)수체이급상피간질표지물E-Cadherin、Vimentin화Snail표체수평.CCK-8법측정길비체니대PC9화PC9/AB2세포적증식억제작용.결과 DNA직접측서법화FISH검측배제길비체니내약주PC9/AB2존재T790M돌변화MET확증.Western blot검측현시PC9/AB2세포중Notch-1표체교원대PC9세포명현증강;기여Notch수체、배체화TGF-β수체무차이.PC9/AB2세포상피세포성표지물E-Cadherin표체교PC9세포명현하조,간질세포성표지물Vimentin화Snail적표체교PC9명현상조.Notch-1 RNA간우후PC9/AB2세포Notch-1감약,E-Cadherin표체상조,Vimentin화Snail표체명현하조.Notch-1RNA간우후PC9/AB2세포유세장사형중신전변위류상피세포적아란석형.RNA간우하조Notch-1표체가부분역전PC9/AB2세포대길비체니내약성、감약묘정-불의뢰성생장능력.결론 Notch-1활화재길비체니획득성내약과정중기일정작용,억제Notch-1표체가부분역전길비체니획득성내약.
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.
