基因沉默脂肪酸合酶下调程序性死亡因子受体配体1增强非小细胞肺癌放疗敏感性  

Gene silence of fatty acid synthase can enhance the radiation sensitivity in non-small cell lung cancer via down-regulation of programmed death receptor ligand 1

作　　者：沈明敬[1] 刘沙沙[2] 徐中华[1] 徐卫华[1] 蒋堪秋[1] 丁启峰 邢鹏飞[3] 陈勇兵[1] 徐忠恒[1] Shen Mingjing;Liu Shasha;Xu Zhonghua;Xu Weihua;Jiang Kanqiu;Ding Qifeng;Xing Pengfei;Chen Yongbing;Xu Zhonghen(Department of Thoracic and Cardiac Surgery,the Second Affiliated Hospital of Soochow University,Suzhou 215004,China;Department of Rehabilitation Medicine,First Affiliated Hospital of Soochow University,Suzhou 215006,China;Department of Radiation Oncology,the Second Affiliated Hospital of Soochow University,Suzhou 215004,China)

机构地区：[1]苏州大学附属第二医院胸心外科,215004 [2]苏州大学附属第一医院快速康复外科、康复医学科,215006 [3]苏州大学附属第二医院放疗科,215004

出　　处：《中华实验外科杂志》2020年第9期1669-1672,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81802989);省部共建放射医学与辐射防护国家重点实验室开放课题(GZK1201904);苏州市民生科技基金(SYS2018064);苏州市重点学科(SZXK201803);苏州市胸部肿瘤研究重点实验室(SZS201907);医院内留学归国博士基金(SDFEYBS1709)。

摘　　要：目的探讨脂肪酸合酶在非小细胞肺癌(NSCLC)中的表达以及调控放疗敏感性的作用与机制。方法选取2015年5月至2018年12月苏州大学附属第二医院NSCLC临床标本,免疫组织化学(IHC)检测肺癌组织脂肪酸合酶(FASN)蛋白表达。以A549、H226为研究对象,采取小剂量累积放疗方法培养相应的放疗耐受株A549R、H226R。蛋白电泳和油红染色分别检测细胞株FASN和脂肪的表达。构建FASN基因沉默的A549R-siFASN、H226R-siFASN后,12 h抽提RNA,24 h抽提蛋白质,经蛋白质印迹法(Western blot)和实时定量反转录聚合酶链反应(RT-qPCR)检测基因沉默组和对照组细胞FASN表达,经单细胞克隆实验检测细胞对放疗的敏感性变化。最后,经免疫荧光检测A549R-siFASN、H226R-siFASN及相应对照组程序性死亡因子受体配体-1(PD-L1)表达。组间比较采用t检验。结果与癌旁组织比较,FASN蛋白在肺癌组织中显著高表达[(13.20±1.83)个/视野比(29.40±3.77)个/视野,t=3.869,P<0.05]。与放疗敏感肺癌组织比较,FASN蛋白在放疗不敏感肺癌组织中显著高表达[(25.90±3.43)个/视野比(39.50±5.06)个/视野,t=2.225,P<0.05];与亲本细胞株A549P、H226P比较,FASN蛋白及脂肪颗粒在放疗耐受株A549R、H226R中表达显著上升[(2.38±0.60)个/视野比(5.88±0.81)个/视野,t=3.477,P<0.05;(4.25±0.53)个/视野比(7.50±0.76)个/视野,t=3.529,P<0.05];与对照组A549R-sc、H226R-sc比较,FASN基因沉默的A549R-siFASN[mRNA相对表达量1.00±0.11比0.35±0.08,t=12.074,P<0.05]和H226R-siFASN[mRNA相对表达量1.00±0.09比0.25±0.01,t=14.653,P<0.05]对放疗的敏感性显著上升,4、6 Gy时A549R-sc/siFASN存活指数为0.82±0.15比0.35±0.07、0.69±0.09比0.3±0.05(t=3.127、6.652,P<0.05),2、4、6 Gy时H226R-sc/siFASN存活指数为(0.77±0.11比0.51±0.12、0.65±0.08比0.3±0.1、0.53±0.07比0.22±0.04,t=6.887、9.571、10.680,P<0.05)。与对照组A549R-sc、H226R-sc比较,FASN基因沉默的A549R-siFASN、H226R-siFASN中PD-L1表Objective To investigate the regulation role of fatty acid synthase(FASN)in radiation sensitivity and its associated mechanism in non-small cell lung carcinoma(NSCLC).Methods Sixty-five cases of NSCLC tumor tissues and corresponding adjacent tissues from radiation sensitive patients or radiation insensitive patients from the second affiliated hospital of Soochow university from may 2015 to December 2018 were collected.FASN expression levels were detected by immunohistochemistry(IHC).Radiation resistant cell lines were cultured by accumulation of low dose radiation therapy.RNA was extracted in 12 h and protein was extracted in 24 h after FASN knocking down cell lines were constructed.FASN expression was detected in parental cell lines A549/H226 and corresponding radiation resistant cell lines A549R/H226R by Western blotting.Fat expression levels were detected by red oil staining assay.Furthermore,FASN expression levels were detected to confirm the FASN knocking down effect in A549R-siFASN/H226R-siFASN cells by Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).Then,the radiation sensitivity was observed by cell clone formation assay.At last,programmed death receptor ligand 1(PD-L1)expression in A549R-siFASN/H226R-siFASN cells and corresponding control cells was detected by immunofluorescence assay.We use student’s t test for statistical analysis.Results FASN expression increased in NSCLC tumor tissues significantly by comparison of corresponding adjacent tissues.The positive cells in each scope sight is 13.20±1.83 and 29.40±3.77,respectively(t=3.869,P<0.05).FASN expression also increased in radiation insensitive NSCLC tumor tissues significantly by comparison of radiation sensitive tissues.The positive cells in each scope sight is 25.90±3.43 and 39.50±5.06,respectively(t=2.225,P<0.05).The fatty granule of A549P and A549R in each scope sight is 2.38±0.60 and 5.88±0.81,respectively(t=3.477,P<0.05).The fatty granule of H226P and H226R is 4.25±0.53 and 7.50±0

关 键 词：非小细胞肺癌 放疗敏感性 脂肪酸合酶 程序性死亡因子受体配体-1 

分 类 号：R734.2[医药卫生—肿瘤]