克尔斯滕大鼠肉瘤病毒致癌基因同源物突变亚型对非小细胞肺癌细胞程序性细胞死亡配体1和2的表达影响  

Effects of mutation subtypes of kirsten rat sarcoma viral oncogene homolog on the expression of programmed cell death ligands 1 and 2 in non-small cell lung cancer cells

作　　者：白悦 朱公建 郎丽丽 曹群[2] 张逊[3] 孙大强[3] Bai Yue;Zhu Gongjian;Lang Lili;Cao Qun;Zhang Xun;Sun Daqiang(Graduate School of Tianjin Medical University,Tianjin 300070,China;Department of Thoracic Surgery,Gansu Cancer Hospital,Lanzhou 730050,China;Department of Thoracic Surgery,Tianjin Chest Hospital,Tianjin 300110,China)

机构地区：[1]天津医科大学研究生院,300070 [2]甘肃省肿瘤医院胸外科,730050 [3]天津市胸科医院胸外科,300222

出　　处：《中华实验外科杂志》2021年第8期1427-1430,共4页Chinese Journal of Experimental Surgery

基　　金：甘肃省卫生健康行业科研项目(GSWSKY2020-40)。

摘　　要：目的探讨克尔斯滕大鼠肉瘤病毒致癌基因同源物(KRAS)突变亚型对非小细胞肺癌细胞程序性细胞死亡配体1(PD-L1)和2(PD-L2)的表达影响。方法纳入2018年1月至2021年1月甘肃省肿瘤医院收治的KRAS阳性非小细胞肺癌患者95例,通过测序检测KRAS基因组序列,通过实时荧光定量聚合酶链反应(PCR)和免疫印迹检测PD-L1和PD-L2的表达。使用成簇的规则间隔的短回文重复基因编辑(CRISPR/CAS9)技术构建KRAS缺失型A549细胞,在敲除株中过表达KRAS野生型和突变型,检测其对PD-L1和PD-L2的表达影响。将KRAS野生型和突变型过表达的KRAS敲除人肺泡上皮细胞549(A549)细胞株与树突状细胞和细胞因子诱导的杀伤细胞(DC-CIK),通过流式细胞术检测群集行列式蛋白3阳性(CD3^(+))细胞的凋亡水平。使用t检验比较连续变量。使用Mann-Whitney检验分析KRAS突变型患者PD-L1和PD-L2。Chi-square检验分析非小细胞肺癌患者KRAS突变型和KRAS野生型的临床特征。结果纳入KRAS阳性非小细胞肺癌患者95例,KRAS野生型31例、KRAS突变型64例;其中KRAS第12位甘氨酸突变为天冬氨酸(G12D)13例、突变为缬氨酸(G12V)12例、突变为半胱氨酸(G12C)25例、突变为丙氨酸(G12A)14例。KRAS突变型患者PD-L1和PD-L2的mRNA表达水平(1.062比1.834,U=38;1.062比1.668,U=70;1.062比1.544,U=111;1.062比1.479,U=89,P<0.05;1.067比1.798,U=68;1.067比1.595,U=86;1.067比1.527,U=171;1.067比1.680,U=34,P<0.05)和蛋白表达水平均高于KRAS野生型患者,差异有统计学意义。在KRAS敲除A549细胞株中,相较于KRAS野生型,KRAS突变型更能够促进PD-1配体PD-L1和PD-L2的表达同时,将KRAS-G12D,KRAS-G12V,KRAS-G12C,KRAS-G12A质粒混合在一起作为RAS野生型(KRAS-MT)与KRAS突变型(KRAS-WT)分别转染KRAS敲除A549细胞株,KRAS-MT依旧可以促进PD-1配体PD-L1和PD-L2的表达。在KRAS敲除A549细胞株中过表达KRAS-MT与KRAS-WT后,KRAS突变亚型不能激活细胞外信号调节激酶(ERK)通路,Objective To investigate the effect of Kirsten rat sarcoma virus oncogene homolog(KRAS)mutation subtype on the expression of programmed cell death ligand 1(PD-L1)and programmed cell death ligand 2(PD-L2)in non-small cell lung cancer cells.Methods A total of 95 KRAS-positive non-small cell lung cancer patients who were admitted to Gansu Cancer Hospital from January 2018 to January 2021 were enrolled.KRAS genome sequence was detected by sequencing,and the expression of PD-L1 and PD-L2 was detected by real-time fluorescent quantitative polymerase chain reaction(PCR)and western blotting.The clustered regularly interspaced short palindrome repetitive gene editing(CRISPR/CAS9)technology were used to construct KRAS-deficient A549 cells.The KRAS wild type and mutant were overexpressed in knockout strain,and its effect on PD-L1 and PD-L2 Expression were detected.The wild-type and mutant KRAS overexpressed KRAS knockout human alveolar epithelial cell line 549(A549)was compared with dendritic cells and cytokine induced killer cells(DC-CIK).The apoptotic level of cluster determinant 3 positive(CD3^(+))cells was detected by flow cytometry.The t test was used to compare continuous variables.The Mann-Whitney test was used to analyze the PD-L1 and PD-L2 of KRAS mutant patients.The chi-square test was used to analyze the clinical characteristics of KRAS mutant and KRAS wild-type in patients with non-small cell lung cancer.Results 95 cases of KRAS-positive non-small cell lung cancer patients,31 cases of KRAS wild type,and 64 cases of KRAS mutant type were included.Among them,13 cases of KRAS glycine mutation were mutated to aspartic acid(G12D),12 cases were mutated to valine(G12V),25 cases were mutated to cysteine(G12C),and 14 cases were mutated to alanine(G12A).The mRNA expression levels of PD-L1 and PD-L2 in patients with KRAS mutation and protein expression levels were higher than those of KRAS wild type patients(1.062 vs.1.834,U=38;1.062 vs.1.668,U=70;1.062 vs.1.544,U=111;1.062 vs.1.479,U=89,P<0.05;1.067 vs.1.798,U=68;1.067 vs

关 键 词：肺癌 突变 非小细胞程序性细胞死亡配体1 程序性细胞死亡配体2 

分 类 号：R734.2[医药卫生—肿瘤]