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Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro
Shen Hui, Ou Haibin, Shao Jin, Jiang Yaofei, Liu Yu, Zhang Junhong, 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
AbstractObjective 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-H2AX 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 D0 value was decreased from 1.28Gy to 1.03Gy, SF2decreased from 0.29 to 0.21, and the sensitization ratio was 1.24). The γ-H2AX focus formation test showed that the number of damage focus formed at 1h and 6h 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.
Fund:National Natural Science Foundation of China (81001099,81370070);Science and Technology Innovation Cultivation Fund of Zhongnan Hospital of Wuhan University (cxpy2017027);Medical Science and Technology Innovation Platform Support Project of Zhongnan Hospital of Wuhan University (PTXM2019030)
Corresponding Authors:
Liu Yu, Email:liuyu97@whu.edu.cn
Cite this article:
Shen Hui,Ou Haibin,Shao Jin et al. Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro[J]. Chinese Journal of Radiation Oncology, 2021, 30(9): 949-955.
Shen Hui,Ou Haibin,Shao Jin et al. Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro[J]. Chinese Journal of Radiation Oncology, 2021, 30(9): 949-955.
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