BCAR1促进肺腺癌细胞增殖、生长及其互作蛋白网络的研究

Promoting effect of breast cancer anti-estrogen resistance 1 on proliferation and growth of lung adenocarcinoma cells and potential networks of its interacting proteins

目的本研究旨在探索乳腺癌抗雌激素药物耐药蛋白1(breast cancer anti-estrogen resistance 1,BCAR1,p130cas)在肺癌发生发展中的生物功能与互作蛋白网络。方法(1)免疫印迹实验(western blot)研究15对肺癌及癌旁组织中BCAR1表达情况,免疫组化(immunohistochemistry,IHC)分析54例早期肺腺癌组织芯片中BCAR1与预后的关系,并通过TCGA数据库进一步验证。(2)通过CRISPR/Cas9分别构建人肺腺癌H1975,H1299 BCAR1敲除(knock out,KO)细胞株,通过调取cDNA文库构建A549过表达(overexpression,OE)细胞株,四唑盐比色实验(MTT)检测细胞增殖能力、平板克隆形成实验检测细胞存活能力。(3)免疫共沉淀质谱分析(immunoprecipitation mass spectrometry,IP-MS)联合生信分析找出工具细胞即293T细胞中BCAR1的互作蛋白网络及可能的关键节点。(4)western blot验证细胞系BCAR1-KO或BCAR1-OE后关键节点基因的表达变化,并在肺癌组织芯片中予以验证基因表达的相关性。结果(1)western blot:BCAR1在肺腺癌组织较癌旁组织显著高表达(P<0.05);IHC:BCAR1高表达的早期肺癌患者预后差(HR=5.026,P=0.001),TCGA:BCAR1高表达肺腺癌患者预后差(HR=1.5,P=0.0087)。(2)成功构建BCAR1-KO与BCAR1-OE稳定细胞株,细胞增殖能力及细胞克隆形成率均随BCAR1敲除降低,过表达后升高。(3)IP-MS联合生信分析:哺乳动物酵母同源致命因子Sec13蛋白8(MLST8)可能作为BCAR1的关键互作蛋白;(4)相比癌旁组织,MLST8在癌组织中高表达(P<0.05),并与BCAR1表达显著正相关(R=0.4221,P=0.0015)。细胞系中MLST8的表达在BCAR1敲除后降低,过表达后升高。结论BCAR1在肺癌中发挥重要的促瘤效应。MLST8表达与BCAR1显著相关,可能是BCAR1的关键下游蛋白。

Objective To explore the biological functions of breast cancer anti-estrogen resistance 1(BCAR1,also known as p130 cas)in the incidence and development of lung cancer,and investigate the potential networks of its interacting proteins.Methods(1)Western blotting was performed to study the expression of BCAR1 in 15 pairs of lung cancer tissues and para-cancerous normal tissues,and immunohistochemical(IHC)assay was carried out in paraffin-embedded tissues from 54 patients with early stage lung cancer in order to determine the relationship between BCAR1 level and prognosis.TCGA analysis was also performed as further validation.(2)BCAR1 knockout(KO)cell lines were established in H1975 and H1299 lung cancer cells,respectively,using CRISPR/Cas9.And A549 cells with overexpression(OE)of BCAR1 were constructed by transfection of BCAR1 cDNA.MTT analysis and plate colony formation assay were used to detect the proliferation and viability of the cells,respectively.(3)Immunoprecipitation mass spectrometry(IP-MS)along with bioinformatics analysis was employed to identify the key protein of BCAR1 interacting networks in 293 T tool cells.(4)The expression variations of the key node gene were confirmed in BCAR1-KO and BCAR1-OE cells using Western blotting,and the correlation between the key node gene and BCAR1 was verified in the lung cancer tissue microarray.Results(1)The expression of BCAR1 was significantly higher in the lung cancer tissues than para-cancerous tissues(P<0.05).Higher expression of BCAR1 was associated with worse prognosis in the patients with early-stage lung cancer(HR=5.026,P=0.001)and TCGA cohort study(HR=1.5,P=0.0087).(2)BCAR1-KO and BCAR1-OE cells were successfully constructed,and the cell proliferation and colony formation were decreased with BCAR1-KO and increased following BCAR1-OE respectively.(3)Mammalian lethal with SEC13 protein 8(GβL,mLST8)was revealed to be a key protein of BCAR1 interacting network.(4)MLST8 was significantly expressed at a higher level in the lung cancer tissues than the para-cancerous tissues(P<0.05),and its expression was positively correlated with the expression of BCAR1(R=0.4221,P=0.0015).Furthermore,the expression of MLST8 was decreased in BCAR1-KO cells and increased in BCAR1-OE cells.Conclusion BCAR1 plays a critical carcinogenetic role in lung cancer.MLST8 expression is significantly correlated with BCAR1,and may be a key downstream regulatory protein of BCAR1.