Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro
10.3760/cma.j.cn113030-20200209-00051
- VernacularTitle:Myosin X对肺癌H1975细胞系放射敏感性调节及机制探讨
- Author:
Hui SHEN
1
;
Haibin OU
;
Jin SHAO
;
Yaofei JIANG
;
Yu LIU
;
Junhong ZHANG
;
Conghua XIE
Author Information
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
- Keywords:
Myosin X;
CRISPR/Cas9 technique;
Radiosensitivity;
H1975 cell line
- From:
Chinese Journal of Radiation Oncology
2021;30(9):949-955
- CountryChina
- Language:Chinese
-
Abstract:
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 2AX 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). The γ-H 2AX 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.
