The Role And Mechanism Of Autophagy In Invasion And Metastasis Of Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma is one of the most common types of malignant tumors in the world,with a high degree of malignancy and rapid disease progression.It has become the fourth leading cause of cancer death worldwide,with high morbidity and mortality annually.Autophagy is a genetically regulated,finely coordinated process of selective cell survival and cell apoptosis,capable of degrading and recycling unnecessary and dysfunctional cellular components.Autophagy occurs in a variety of malignant tumour cells and plays an important role in tumour development.So it is important to study the relationship between autophagy and tumours.In this study,the autophagic activity of hepatocellular carcinoma cells was altered by the autophagy inhibitor chloroquine(CQ).The effects of altered autophagy level on the process of epithelial to mesenchymal transition(EMT)and the migration and invasion ability of cells were observed and the related mechanism was investigated.Methods:Western blotting assay was performed to detect the basal expression of EMT related proteins in hepatocellular carcinoma cell lines.The CCK-8 experiment was used to observe the ability of CQ to inhibit the proliferation of Hep3 B cells under different drug concentrations(10 μM,20 μM,40 μM)and different treatment times(24 h,36 h,48 h).Hep3 B cells were treated with different drug concentrations of CQ(10 μM,20 μM and 40μM)and the change in the expression levels of autophagy related proteins LC3-II/LC3-I,P62,Beclin 1,Atg 5 and EMT related proteins E-Cadherin,Vimentin,N-Cadherin were detected after 24 h.At the same time,Hep3 B cells were treated with CQ 20 μM and the change in the expression levels of autophagy related proteins and EMT related proteins were detected after 24 h,36 h and 48 h respectively.Finally,Wound healing assay and Transwell experiment were conducted to observe the change of migration and invasion ability of Hep3 B cells after treated with 20 μM CQ for 24 h.Results:CQ could significantly inhibit the proliferation of Hep3 B cells in a concentration and time dependent manner.Compared with the control group,the expression of autophagy marker molecule LC3-II/LC3-I was elevated in Hep3 B cells after CQ treatment,accompanied by the accumulation of P62,while the expression of Beclin 1 and Atg 5 was down-regulated.In addition,the expression level of epithelial phenotype marker E-Cadherin was increased,whereas the expression levels of mesenchymal phenotype markers Vimentin and N-Cadherin were decreased.Finally,Wound healing assay and Transwell assay revealed that the migration and invasion ability of Hep3 B cells were diminished when the autophagy activity was inhibited by CQ.Conclusions:CQ inhibited autophagy of hepatocellular carcinoma cells and at the same time prevented the EMT process,causing up-regulation of epithelial phenotype protein and down-regulation of mesenchymal phenotype proteins,and significantly inhibited the proliferation,migration and invasion of hepatocellular carcinoma cells.Autophagy may play an important role in the migration and invasion of hepatocellular carcinoma by affecting EMT process.The targeted regulation of autophagy and EMT signal transduction may prevent the invasion and metastasis of hepatocellular carcinoma cells.Therefore,the inhibition of autophagy may represent a new target for therapeutic intervention.