神经纤维瘤蛋白1在胆囊癌中的机制研究

Mechanism study of neurofibromin 1 in gallbladder cancer

目的探讨神经纤维瘤蛋白1(NF1)在胆囊癌中的机制。方法采用实验研究方法。培养人胆囊癌细胞系GBC‑SD、NOZ、SGC996、EH‑GB1、ZJU0428,人胚肾细胞系293T,人宫颈癌细胞系HELA;构建免疫共沉淀实验所需的重组质粒mRFP‑YAP1^(FL)‑FLAG和eGFP‑MYC‑NF1^(2650~2750)‑HA,体外纯化Yes相关蛋白1(YAP1)截断体蛋白和NF1融合蛋白。分别采用等温滴定量热实验、GST pull‑down实验、免疫共沉淀实验、免疫荧光和激光共聚焦检测NF1与YAP1的体内外相互作用,采用蛋白质免疫印迹法检测不同胆囊癌细胞系中NF1蛋白的表达水平。观察指标:(1)NF1与YAP1的体外相互作用。(2)NF1与YAP1的细胞内相互作用。(3)不同人胆囊癌细胞系中NF1蛋白的表达水平。结合解离常数由ITC200等温滴定量热仪自带软件输出,以x±s表示。计数资料以绝对值表示。结果(1)NF1与YAP1的体外相互作用。①等温滴定量热实验结果显示:PPQY肽段滴定体外纯化YAP1的第1个WW1结构域蛋白存在相互作用,结合解离常数为(0.42±0.06)mmol/L;PPQY肽段滴定体外纯化YAP1的162~275蛋白区段存在相互作用,结合解离常数为(0.69±0.14)mmol/L。②GST pull‑down实验结果显示:相对于GST蛋白和His‑Sumo‑YAP1 WW1反应体系,在GST‑PPQY融合蛋白与His‑Sumo‑YAP1^(WW1)反应体系的蛋白泳道中可以观察到目的蛋白His‑Sumo‑YAP1^(WW1);相对于GST蛋白和His‑Sumo‑YAP1^(WW2)反应体系,在GST‑PPQY融合蛋白与His‑Sumo‑YAP1^(WW2)反应体系的蛋白泳道中可以观察到目的蛋白His‑Sumo‑YAP1^(WW2)。(2)NF1与YAP1的细胞内相互作用。①免疫共沉淀实验结果显示:在使用FLAG凝胶珠孵育共转染mRFP‑YAP1 FL‑FLAG和eGFP‑MYC‑NF12650~2750‑HA的细胞裂解液中,观察到NF1蛋白的免疫印迹。②免疫荧光和激光共聚焦检测结果显示:在人胆囊癌细胞系NOZ的原位荧光中,YAP1和NF1荧光明显,主要定位于细胞质中。在人胆囊癌细胞系SGC996的原位荧光中,YAP1荧光明显,主要定位于细胞核中,NF1荧光不明显。(3)不同人胆囊癌细胞系中NF1蛋白的表达水平。蛋白印迹实验结果显示:以人宫颈癌细胞系HELA中NF1蛋白的表达量为标准,在人胆囊癌细胞系EH‑GB1、GBC‑SD、NOZ、SGC996、ZJU0428中,NF1蛋白的相对表达量分别为1.28、0、1.01、0、0。结论NF1可能通过直接作用于YAP1蛋白影响胆囊癌。

Objective To investigate the mechanism of neurofibromin 1(NF1)in gallbla-dder cancer.Methods The experimental study was conducted.Human gallbladder cancer cell lines,including GBC-SD,NOZ,SGC996,EH-GB1,ZJU0428,human embryonic kidneys cell line 293T and human cervical cancer cell line HELA,were cultured.The recombinant plasmids(mRFP-YAP1 FL-FLAG aneGFP-MYC-NF1^(2650‒2750)-HA)were constructed for co-immunoprecipitation experiment truncated Yes associated protein 1(YAP1)and NF1 recombinant proteins were purified in vitro.The interaction between NF1 and YAP1 in vitro or in vivo were verified by isothermal titration calori-metry(ITC)assay,GST pull-down experiment,co-immunoprecipitation,immunofluorescence,laser confocal microscopy,and the expression of NF1 protein in different gallbladder cancer cell lines was verified by Western blot experiments.Observation indicators:(1)interaction between NF1 and YAP1 in vitro;(2)interaction between NF1 and YAP1 in cells;(3)expression of NF1 protein in different human gallbladder cancer cell lines.The dissociation constants were exported from ITC 200 software and represented as Mean±SD.Count data were represented as absolute numbers.Results(1)Interaction between NF1 and YAP1 in vitro.①Results of ITC assay showed that there was interac-tion between PPQY and YAP1-WW1,between PPQY and YAP1(Amino acid residues 162‒275),and the dissociation constants between PPQY and YAP1-WW1,between PPQY and YAP1(Amino acid idues 162‒275)were(0.42±0.06)mmol/L,(0.69±0.14)mmol/L,respectively.②GST pull-down esults indicated that the target protein His-Sumo-YAP1^(WW1) was obviouly observed in protein lane of eaction system between GST-PPQY recombinant protein and His-Sumo-YAP1^(WW1),relative to the reaction system between GST protein and His-Sumo-YAP1^(WW1).The target protein His-Sumo-YAP1^(WW2) was obviouly observed in protein lane of tion system between GST-PPQY recombinant protein and His-Sumo-YAP1^(WW2),relative to the reaction system between GST protein and His-Sumo-YAP1^(WW2).(2)Interaction between NF1 and YAP1 in cells.①Co-immunoprecipitation results indica-ted that NF1 protein was observed in cell lysis solution which was incubated by FLAG gel beads cotransfected with mRFP-YAP1 FL-FLAG and eGFP-MYC-NF12650‒2750-HA.②Immuno-fluorescence and laser confocal microscopy results indicated that YAP1 and NF1 with obvious fluorescence were co-localized in the cytoplasm of human gallbladder cancer NOZ cells.However,YAP1 with obvious e was localized in the nucleus of human gallbladder SGC996 cells and NF1 showed weak fluorescence.(3)Expression of NF1 protein in different human gallbladder cancer cell lines.Western blot results showed that with the expression level of NF1 protein in HELA cell line as the standard,the relative expression levels of NF1 protein in EH-GB1,GBC-SD,NOZ,SGC996,ZJU0428 cell lines were 1.28,0,1.01,0,0,respectively.Conclusion NF1 affects the gallbladder cancer by directly acting on YAP1 protein.