Study On Targeting Treatment Of Hepatocellular Carcinoma Induced By Autophagy And Autophagy In Liver

Objective:1. To investigate autophagy phenomenon innormal liver and hepatocellular carcinoma cells.2.To investigate the level of autophagy variation of the liver during ischemia-reperfusion injury.3.To observewhether preconditioning effect is accompanied with changes in the level of autophagy.4. To explore the molecules and signaling pathways involved in the crosstalk between ischemic preconditioning and autophagy.5.To explore the relationship between autophagy regulation and chemo sensitivity of hepatocellular carcinoma cells.Methods:1. Build C57BL6mice70%warm ischemia-reperfusion model, compare the ALT levels, cell necrosis and ATP levels of normal group, ischemia reperfusion group, and ischemic preconditioning group.2. Using Western Blot to detect autophagy specific marker molecules LC3protein, and transmission electron microscopyto detect autophagosome, compare the trend of autophagy level at different time points of the three groups.3.Using Western Blot to detect P70S6K and4EBP1protein to investigate the activity of mTORCl complex.4.Using Western Blot to detect the expression of nuclear transcription factor TFEB, and to explore the possible molecular signaling pathways.5.Using software to predict specific target miRNA of autophagy gene STMN1, RAB5A, ATG4D and mTOR, and then HepG2cells were transfected with miRNA mimics of specific target function.6.Four target gene (STMN1, RAB5A, ATG4D and mTOR) plasmids containing matching or mutant sequences of each gene were constructed respectively.7.Using Western Blot and transmission electron microscopy to estimate the level of autophagy of HepG2cells transfected with specific target miRNA mimics or inhibitor.8.Using flow cytometry to detect the apoptosis of different groups of HepG2cells treated with cisplatinResults:1. Ischemic preconditioning can cause a decreaseof serum ALT levels and cell necrosis and increase the ATP level of the mice live.2. Western Blot and transmission electron microscopy confirmed ischemic preconditioning can enhance liver autophagy.3.Ischemic preconditioning can cause the mTORC1complex activity and TFEBnuclear translocationin the mice liver.4.miR-101significantly reduced the activity of the STMN1, RAB5A, ATG4D and mTOR3’-UTR luciferase plasmids, while plasmids containing mutant sequences were not significantly affected.5. q-PCR and Western Blotconformed that the mRNA and protein levelsof STMN1, RAB5A, ATG4D, and mTOR were downregulated by miR-101.6. Transmission electron microscopy and Western Blot confirmed suppression of autophagy by miR-101.7. HepG2cells with cisplatin pre-treatment for48hours, the group transfected with miR-101-mimic the apoptotic ratio of25.37±4.05%, while the miR-101-mimic-control group was only14.28±1.73%; and the apoptosis ratio of miR-101-inhibitor group was7.35±2.25%, while the inhibitor-control group was15.31±2.31%; wild type HepG2cells was15.19±2.37%.Conclusions:1. There is a baseline level of autophagy in both normal liver and hepatocellular carcinoma cells.2.Autophagy was activated during hepatic ischemiareperfusion.3. Ischemic preconditioning can ease the hepatic ischemia-reperfusion injury.4. The mechanism of ischemic preconditioning may possiblly by inhibiting mTORCl complex activity, promoting TFEB nuclear translocation and activation of autophagy gene.5. miR-101can directly target STMN1, RAB5A, ATG4D, and mTOR and affect both mRNA and protein levels. As a result it plays the function of inhibition of autophagy.6. miR-101-induced autophagy inhibition can enhance the sensitivity of hepatocellular carcinoma cells to cisplatin chemotherapy.7. For hepatic ischemia-reperfusion injury, pre-upregulationof the level of autophagy to reduce the subsequent reperfusion injury may be an idea for the future treatment.8. Gene therapy targeting autophagy should be investigated further as a potential alternative therapeuticstrategy for HCC...