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.
ABSTRACT
Objective To investigate whether the apoptosis-2 ligand (Apo-2L),known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),could enhance irradiation-induced apoptosis in lung adenocarcinima H1975 cells that are resistant to the epithelial growth factor receptor (EGFR)-TKI.Methods Adenocarcinima H1975 cells were randomly divided into four groups:the control group,Apo2L group,irradiation group,and both Apo-2L and irradiation group.H1975 cells were pretreated with Apo2L under different concentrations of 200 and 228 ng/ml at 24 h before irradiation with doses of 1,1.5,2,2.5,3,3.5 and 4 Gy.The apoptosis rates of all groups were analyzed by flow cytometry 24 h post-irradiation.The inhibition rates of cell proliferation were measured by the MTT assay.Results MTT assay showed that the Apo-2L treatment significantly inhibited cell proliferation(x2 =136.17,P ＜ 0.05).The apoptosis rates of the four groups were different significantly,and the apoptosis rate of radiation combined with drug group was significantly higher than the other three groups(x2 =78.02,P ＜0.05).Conclusions The Apo-2L could not only inhibit the proliferation but also promote radiation-induced apoptosis of adenocarcinoma H1975 cells.