Study On The Autophagy And The Relationship Between Autophagy And Apoptosis Induced By Inactivated Sendai Virus Strain Tianjin In Human Cervical Carcinoma HeLa Cells

Objective: Oncolytic virus therapy is considered to be one of the most promising cancer treatments in recent years.Studies have shown that a variety of oncolytic viruses exert anti-tumor effects in vitro or in vivo by inducing specific apoptosis or autophagy of tumor cells and anti-tumor immunity.In the previous work,our team found that Sendai virus strain Tianjin isolated in our laboratory can induce apoptosis of human cervical carcinoma HeLa cells after inactivation by ultraviolet rays.This study intends to further examine the ultraviolet-inactivated Sendai virus strain Tianjin(hereinafter referred to as UV-Tianjin)to induce HeLa cell autophagy and its preliminary mechanism,the relationship between HeLa cell autophagy and apoptosis,and the role of reactive oxygen species(ROS)in autophagy and apoptosis induced by UV-Tianjin,thus laying a theoretical and experimental basis for the study of the antitumor mechanism of Sendai virus.Methods:(1)First,we studied the occurrence of autophagy induced by UV-Tianjin and its preliminary mechanism.HeLa cells were transfected with the GFP-LC3 plasmid,and then infected with different titers of UV-Tianjin.A confocal microscope or a fluorescent microscope was used to observe Hela cells produce autophagy.Western Blot was used to detect the expression of autophagy marker LC3 and the levels of phosphorylation of PI3 K,Akt and m TOR in the classic autophagy pathway PI3 K / Akt / m TOR detects in Hela cells infected with different titers of UV-Tianjin.(2)Next,we explored the relationship between UV-Tianjin-induced autophagy and apoptosis in HeLa cells.(1)HeLa cells pretreated with autophagy inhibitor 3-MA were infected with UV-Tianjin(MOI:80),Flow cytometry was used to detect the apoptotic rate.Western Blot was used to detect the expression levels of PARP and caspase-3 in HeLa cells.(2)HeLa cells pretreated with autophagy inhibitor Z-VAD-FMK were infected with UV-Tianjin(MOI:80),and the autophagy production was observed by a fluorescence microscope.The expression level of LC3 in the cells was detected by Western Blot.(3)Finally,we explored the role of ROS in UV-Tianjin-induced autophagy and apoptosis.(1)Fluorescence microscope and flow cytometry were used to detect the level of ROS production in HeLa cells infectedwith UV-Tianjin at different titers.(2)HeLa cells pretreated with antioxidant NAC were infected with UV-Tianjin(MOI:80),and Flow cytometry was used to detect ROS generation level.Autophagy production was observed by a fluorescence microscope.Western Blot was used to detect LC3 expression in cells.Flow cytometry was used to detect the apoptotic rate.The expression levels of PARP and caspase-3 in the cells were detected by Western Blot.Results:(1)UV-Tianjin-induced autophagy and its preliminary mechanism: Confocal microscopy observations showed that the red fluorescence dots after the merge of red and green fluorescence were significantly increased,suggesting the formation of autophagy flow.Observation by fluorescence microscope revealed that the number of green fluorescent spots in UV-Tianjin-treated HeLa cells increased significantly and had a dose-dependent trend.Western Blot results showed that with the increase of UV-Tianjin titer,the expression level of LC3-II was up-regulated(P<0.05),while the expression levels of p-m TOR,p-PI3 K and p-Akt were down-regulated(p<0.01).(2)The relationship between autophagy and apoptosis of HeLa cells induced by UV-Tianjin: The results of flow cytometry showed that compared with the virus control group,the apoptosis rate of 3-MA pretreated HeLa cells was significantly increased(p<0.05).Western Blot results showed that the expression levels of cleaved PARP and cleaved caspase-3 in 3-MA pretreated HeLa cells were up-regulated(p<0.05).Fluorescence microscopy showed that compared with the virus control group,green fluorescent spots were significantly increased in z-VAD-fmk pretreated HeLa cells.Western Blot results showed that the expression level of LC3-II was up-regulated in z-VAD-fmk pretreated HeLa cells(p<0.05).(3)The role of ROS in UV-Tianjin-induced autophagy and apoptosis: Fluorescence microscopy and flow cytometry showed that ROS content in UV-Tianjin-infected HeLa cells increased in a dose-dependent manner.Compared with the virus control group,ROS production was significantly inhibited in NAC-treated HeLa cells.Fluorescence microscopy showed that compared with the virus control group,the green fluorescent spots in NAC-treated HeLa cells were significantly reduced.Western Blot results showed that NAC pretreatment the expression of LC3-II was down-regulated in the treated HeLa cells(p<0.01).Flow cytometry results showed that compared with the virus controlgroup,the apoptosis rate of HeLa cells pretreated by NAC was significantly reduced.Western Blot results showed that the expression levels of cleaved PARP and cleaved caspase-3 in NAC pretreated HeLa cells were down-regulated(p<0.05).Conclusion: UV-Tianjin-induced autophagy in HeLa cells was closely associated with the PI3K/Akt/m TOR pathway.The autophagy and apoptosis induced by UV-Tianjin inhibit each other in HeLa cells.ROS plays an important role in UV-Tianjin-induced apoptosis and autophagy.ROS may promote both the autophagy and apoptosis induced by UV-Tianjin in HeLa cells.