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

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.