Myosin X对肺癌H1975细胞系放射敏感性调节及机制探讨  

Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro

作　　者：沈惠 欧海斌 邵晶 江耀飞 刘羽[1] 张俊红[1] 谢丛华[1] Shen Hui;Ou Haibin;Shao Jin;Jiang Yaofei;Liu Yu;Zhang Jun hong;Xie Conghua(Department of Radiation and Medical Oncology,Hubei Key Laboratory of Tumor Biological Behaviors,Hubei Cancer Clinical Study Center,Hubei Cancer Radiation Therapy Quality Control Center,Zhongnan Hospital of Wuhan University,Wuhan 430071,China)

机构地区：[1]武汉大学中南医院放化疗科湖北省肿瘤生物学行为重点实验室湖北省肿瘤医学临床研究中心湖北省肿瘤放疗质量控制中心,430071

出　　处：《中华放射肿瘤学杂志》2021年第9期949-955,共7页Chinese Journal of Radiation Oncology

基　　金：国家自然科学基金项目(81001099,81370070);武汉大学中南医院科技创新培育基金(expy2017027);武汉大学中南医院医学科技创新平台支撑项目(PTXM2019030)。

摘　　要：目的探讨Myosin X对肺癌H1975细胞系放射敏感性调节及相关机制。方法蛋白免疫印迹法检测肺癌细胞系中Myosin X表达量以获取实验对象。运用CRISPR/Cas9技术建立敲除Myosin X的H1975细胞系(KO组)和感染对照病毒的H1975细胞系(NC组),并进行敲除效率验证。克隆形成实验及多靶单击模型检测两组细胞对射线敏感性差异。γ-H_(2)AX焦点形成实验及RNAseq差异分析技术寻找可能参与Myonsin X介导的肺癌细胞系放射敏感性调节机制。结果Myosin X在H1975细胞中表达量明显高于其他细胞系(P<0.01)。经鉴定成功构建了Myosin X sgRNA-Lenti-CRISPR v2慢病毒载体,嘌呤霉素筛选后得到Myosin X完全敲除的H1975(KO组)及对照组(NC组)细胞系。克隆形成实验证明相较于NC组,KO组对射线的敏感性更强(D_(0)值从1.28 Gy下降至1.03 Gy,SF_(2)从0.29下降至0.21,放射敏感性比为1.24)。γ-H_(2)AX焦点形成实验显示照后1、6 h KO组形成的损伤焦点数均多于NC组(P<0.05),RNAseq差异分析技术结果显示KO组ISLR蛋白表达明显低于NC组(P<0.05)。结论敲除Myosin X可以增强肺癌H1975细胞的放射敏感性,其机制可能与干扰DNA双链损伤修复以及降低ISLR表达有关。Objective To investigate the effect and mechanism of Myosin X on the radiosensitivity of non-small cell lung cancer(NSCLC)cell line H1975 in vitro.Methods Western blot was applied to detect the expression level of Myosin X expression.The H1975 cell line with stable knockout of Myosin X(KO group)and infected with control virus(NC group)were constucted by using CRISPR/Cas9 technique.The knockout efficiency was validated.The radiosensitivity of two cell lines was measured by colony formation assay and single-hit multi-target model.γ-H_(2)AX focus formation test and RNA sequencing(RNAseq)analysis were employed to identify the regulatory mechanism of the radiosensitivity of lung cancer cell lines mediated by Myosin X.Results The expression level of Myosin X in the H1975 cells was significantly up-regulated than those in other NSCLC cell lines(all P<0.01).The lentiviral vector of Myosin X sgRNA-Lenti-CRISPR v2 was successfully constructed.After the puromycin screening,H1975 cell lines with complete knockout of Myosin X and control cell lines(NC group)were obtained.Colony formation assay demonstrated that compared with the NC group,the radiosensitivity in the KO group was significantly higher(The D_(0)value was decreased from 1.28 Gy to 1.03 Gy,SF_(2)decreased from 0.29 to 0.21,and the sensitization ratio was 1.24).Theγ-H_(2)AX focus formation test showed that the number of damage focus formed at 1 h and 6 h after irradiation in the KO group was significantly larger than that in the NC group P<0.05.RNAseq analysis indicated that the expression level of ISLR in the KO group was significantly down-regulated than that IN the NC group(P<0.05).Conclusion Knockout of Myosin X can increase the radiosensitivity of H1975 cells probably by interfering the repair of DNA double-strand damage and down-regulating the expression level of ISLR.

关 键 词：肌球蛋白X CRISPR/Cas9技术 放射敏感性 H1975细胞系 

分 类 号：R734.2[医药卫生—肿瘤]