Study On The Apoptosis And Autophagy In The Hepg-2 Cells Inhibited With Apoptin

Background:Primary liver cancer is the second leading cause of cancer death in the world.In January 2018,the American Cancer Society published 2018 cancer statistics and the results show that the total cancer death rate in the United States has dropped by 25%in the past 25 years,and the mortality rate of liver cancer is still rising.According to statistics,about 700,000 new liver cancer patients and 600?000 patients die of liver cancer every year in the world,and more than 300,000 people die of liver cancer every year in China,accounting for about half of the global liver cancer deaths.Hepatocellular carcinoma has a high degree of malignancy and a very poor prognosis.It is still an effective method for the treatment of liver cancer before surgical resection.However,the long-term efficacy is still poor.Therefore,finding effective and low-toxic drugs is one of the effective methods for treating liver cancer.It is generally believed that the occurrence and development of tumors are not only the result of uncontrolled cell proliferation and abnormal differentiation,but also related to the imbalance of tumor cell apoptosis.However,with the development of molecular biology,apoptosis is not the only one that determines the fate of cell death.In recent years,autophagy has been shown to co-regulate cell death with apoptosis.In some cases,autophagy inhibits apoptosis,but autophagy itself induces cell death,or interacts with apoptosis and induces cell death as a backup mechanism in the absence of apoptosis.Apoptin is a protein that specifically induces apoptosis in tumor cells.Since this protein is not mediated by p53 protein and is not inhibited by overexpression of Bcl-2,it is considered a novel anti-tumor biological protein.However,the autophagy activation of apoptin and its interaction with apoptosis have not been precisely elucidated.Objective:In the previous study,we have constructed a recombinant virus Ad-apoptin expressing human apoptin protein with human type 5 adenovirus as a vector,and analyzed the main pathways and self-inhibition effects of apoptin-mediated HepG-2 cell killing.The role of phagocytosis and its interaction with apoptosis provides a theoretical basis for further research and development of apoptin.Research methods:1.The killing inhibition effect of apoptin on human primary liver cancer HepG-2 cells was analyzed by means of crystal violet staining,MTS detection,tumor bearing and metastatic tumor model.2.The main pathway of apoptin inhibition of human primary liver cancer HepG-2 cells were analyzed by Hoechst staining,Annexin V-FITC/PI flow cytometry,qPCR and Western blot,JC-1 staining and TMRM staining.The effects of autophagy on the inhibition of autophagy in human primary liver cancer HepG-2 cells were analyzed by LTR staining,qPCR and Western blot.4.Using the apoptosis inhibitor QVD and the autophagy inhibitor CQ?and the means of staining for apoptosis and autophagy,staining for mitochondria,Annexin V-FITC/PI flow detection,qPCR and Western blot were used to analyze the relationship between apoptosis and autophagy in apoptotic inhibition of human primary liver cancer HepG-2 cells.5.Using reactive oxygen species inhibitor NAC,and the means of ROS detection,Annexin V-FITC/PI flow detection,qPCR and Western blot were used to analyze t he effect of the ROS level on apoptosis and autophagy of primary liver cancer cells.Research result:1.Apoptin has a killing inhibitory effect on human primary liver cancer HepG-2 cells.2.Apoptin mainly inhibits the growth of human primary liver cancer HepG-2 cells by endogenous apoptotic pathway.3.Apoptin inhibits the change of autophagy while inhibiting the growth of HepG-2 cells,and inhibition of autophagy could increase the mortality of HepG-2 cells.4.Inhibition of apoptosis in apoptin-treated HepG-2 cells could significantly reduce the autophagy level of cells,and the overall inhibitory effect is greater than 24 h at 12 h and the effect was most obvious at 48 h;inhibition of autophagy could increase the apoptosis induced by apoptin on HepG2 cells,and the effect was most obvious at 24 h.5.Apoptin significantly affect the changes of reactive oxygen species on HepG-2 cells,which are closely related to apoptosis and autophagy,and the higher the level of reactive oxygen species,the greater the effect of autophagy and apoptosis.;inhibition of reactive oxygen species can effectively reduce the apoptosis effect of HepG-2 cells,and the stronger the apoptosis effect,the more obvious the inhibitory effect;in HepG-2 cells treated with apoptin,inhibition of reactive oxygen species can significantly reduce the autophagy level of cells,and the overall inhibitory effect is greater than 24 h at 12 h and the effect was most obvious at 48 h.Conclusion:Apoptin can significantly inhibit the growth of human primary liver cancer HepG-2 cells,mainly causing the death of HepG-2 cells in apoptotic manner,and triggering autophagy.The role of autophagy is dominated by protective effects,and when the level of apoptosis is at its highest,the apoptotic effect may exceed the threshold of protective effect of autophagy,and it dominates,so autophagy is at the lowest level.Apoptin may induce mitochondrial autophagy while inducing apoptosis of HepG-2 cells.Autophagy and apoptosis of HepG-2 cells resulted in changes in mitochondrial ROS levels.Therefore,this study suggests that ROS can be a key factor in linking autophagy and apoptosis through mitochondria.Innovation points:1.The main pathway of apoptin on HepG2 cell apoptosis was determined.2.The effect of apoptin on autophagy in the process of inhibiting HepG2 cells was clarified.3.The interaction between autophagy and apoptosis of HepG2 cells induced by apoptin was analyzed.4.T he effects of ROS on apoptosis and autophagy of HepG2 cells induced by apoptin were determined.