The Role Of Microfilament Sketeton In NDV Inducing Apoptosis Of Human Lung Cancer Cells

Cell microfilament cytoskeleton is the key structure of supporting cell morphology and promoting cell movement.It is mainly composed of microfilaments,microtubules and intermediate filaments.When microfilaments,microtubules and intermediate filaments are damaged,apoptosis will occur.On the contrary,when cells undergo apoptosis,the presence of apoptotic microtubules and apoptotic bodies affects the invasion and migration of tumor cells.The RhoA/ROCK pathway regulates microfilament skeleton proteins,alters tumor cells morphology and affects invasion and migration processes.A Newcastle Disease Virus HBNU/LSRC/F3 strain was stored in our laboratory.In previous studies,it was found that NDV F3 can cause apoptosis in gastrointestinal tumors.However,it has not been reported whether this apoptotic process is related to the microfilament skeleton.In this study,NDV F3 strain was used to infect NCI-H1299 cells,which belong to non-small cell lung cancer cells,to investigate the effect of NDV F3 on NCI-H1299 cells and the relationship between NDV F3 induced microfilament recombination even apoptosis and RhoA/ROCK pathway.Morphological observation revealed the changes in cell surface structure and ultrastructure after virus infection;CCK-8 assay was employed to evaluate the inhibitory effect of NDV infection on human NCI-H1299 cancer cells with different dose and different time.The apoptosis rate of infected NCI-H1299 cells was detected by flow cytometry;The expression levels of RhoA?ROCK2?F-actin and p-MYPT1 proteins,which are closely related to the microfilament sketeton,were evaluated by western blot analysis after NDV infection for 0h,24 h,36h,48h;Scratch assay and cell migration assay were used to observe the effects of virus infection on migration ability.Common inverted phase contrast microscope and scanning electron microscope images showed that the cells changed their normal morphology and structure to fusion and dead shapes after NDV F3 treatment.The microvilli on the cell surface became short,swollen or even shedded.CCK-8 assay results showed that NDV inhibited the proliferation of NCI-H1299 cells in a time-and dose-dependent manner(p<0.05).We used flow cytometry to detect apoptosis.The results showed that the apoptotic rate was(8.20±2.36)% when NCI-H1299 cells were infected for 12 h.The apoptosis rate was(14.27±1.00)% for 18 h.The apoptotic rate was(18.30±0.56)% for 24 h.Compared with that of the negative control group(0.21±0.11)%,the results were statistically significant(p<0.01).The results indicated that NDV F3 could induce apoptosis of NCI-H1299 cells.The apoptosis rate increased with NDV F3 infection time within 24 h.Western blot results showed that the expression levels of RhoA?ROCK2?F-actin and p-MYPT1 proteins have decreased with NDV infection time(p<0.05).Scratch assay and cell migration assay showed the healing rate of NDV-infected group was slower than that of control group and the infected cell' migration ability was greatly inhibited.NDV F3 induced apoptosis and inhibited the proliferation and invasion of NCI-H1299 cells(p<0.01).The apoptotic mechanism may be related to Rho/ROCK pathway which is closely involved in the destruction of microfilament sketeton.These conclusions provide a theoretical basis and a new direction for the anti-tumor researches on Newcastle Disease Virus oncolytic effects.