RABIF Promotes Invasion And Migration Via RAB10-dependant Lipophagy In Hepatocellular Carcinoma

Objective:Tumor cells adapt to metabolic stress in microenvironment through metabolic reprogramming.The conventional view is that aerobic glycolysis and glutamine catabolism support the progression of tumor cells.With the deepening of research in the field of tumor metabolism,the role of lipid metabolism has been recognized more and more.Lipid metabolism includes lipid anabolism and lipocatabolism,among which lipocatabolism is highly activated in tumors and its role in tumors has been widely studied.The degradation of lipid droplet(LD)in cells is the core step of lipid catabolism.The Fatty acids(FA)produced provide FA oxidation and the biosynthesis of lipid molecules to meet the energy and material requirements of cells under metabolic pressure.The degradation of LD can be divided into two pathways:lipopolysis and lipophagy.Lipopolysis is the degradation of LD by neutral lipases in cells.Lipophagy is the autophagic degradation of LD.The role of liponysis in promoting cancer in tumors has been demonstrated.However,lipophagy,as a type of selective autophagy,plays a background-dependent role in tumors.At present,the role of lipophagy in tumor under different conditions still needs to be further studied.Metabolic stress in the primary tumor microenvironment promotes tumor metastasis.Our previous studies have confirmed this mechanism and found that starvation promotes the invasion and migration of Hepatocellular carcinoma(HCC)through the induction of autophagy.Lipoophagy is a type of selective autophagy,which is activated when HCC cells are in starvation state.However,the role of lipophagy on tumors under starvation condition is still unclear.Therefore,in this study,the role and molecular mechanism of lipophagy on HCC under starvation conditions were explored.The process of lipophagy can be divided into three steps.First,autophagosomes specifically recognize LD.Secondly,autophagosomes encapsulate LD and transport it to the lysosome.Finally,LD is degraded by acid lipase in lysosome.The transport of LD by autophagosomes to lysosomes is the core step of lipophagy,in which membrane transport plays an important role.There are a variety of Rab GTPases on the LD surface,which play a role in regulating the targeting,tethering,movement and fusion of LD membrane.A variety of Rab GTPases have been found to be activated under starvation conditions and play a role in the regulation of lipophagy.Therefore,this study explored the molecular mechanism by which RAB GTPases participate in the regulation of lipophagy under starvation conditions.Methods:1.To clarify the role of lipid metabolism in HCC,Using GEPIA website(http://gepia.cancer-pku.cn/)and Proteinatlas website(https://www.proteinatlas.org/)for cancer genome(The cancer genome atlas,The sequencing sets of HCC patients in the TCGA database were analyzed,and 364 patients were grouped according to the expression of lipid metabolizing enzymes in tumor tissues for survival analysis.2.To verify the effect of starvation-induced lipophagy on the proliferation,invasion,and migration of HCC cells,immunofluorescence confocal assay was used to image the LD and autophagosomes specifically stained in HCC cells.Image J software was used to quantify the LD fluorescence staining area and autophagosome fluorescence staining area,as well as the co-localization percentage of LD and autophagosome,and the kit was used to detect triglyceride and FA levels to evaluate the level of lipophagy activation.CCK-8 assay,Transwell assay,and western blotting assay were used to detect the proliferation,invasion,and migration of HCC cells.3.To eliminate the influence of lipolysis,acid lalistat was used to specifically inhibit lipophagy.4.To explore the mechanism of starvation induced lipophagy promoting lipophagy,invasion and migration of HCC cells,Real-time quantitative reverse transcription PCR(real-time transcription PCR)and western blotting were used to detect the mRNA and protein expressions of RAB IF and RAB 10.ProteinAtlas website was used to compare the high expression ratio of RABIF in pancancer.GSVA was used to predict the biological function of RABIF.The GEPIA website was used to analyze the expression of RABIF in the carcinoma and paracancerous tissues of HCC patients in TCGA and to analyze the relationship between the expression of RABIF in the tissues of HCC patients and prognosis.The downstream proteins of Rabif were analyzed using the String database.The GEPIA website was used to analyze the coexpression of RABIF,RAB1A,RAB10,RAB12,RAB15,and other genes in the cancer tissues of patients with HCC.Results:1.Activation of lipophagy in HCC was induced by starvation.2.Starvationinduced lipophagy promoted proliferation,invasion and migration of HCC cells.3.RABIF was highly expressed in HCC cell lines and is associated with the prognosis of HCC patients.4.The expression of RABIF was related to the invasion ability of HCC cell lines.5.RABIF might be involved in hunger-induced lipophgy.6.RABIF promoted lipophgy of starvation-induced HCC cells.7.RABIF promoted proliferation,invasion and migration of starvation-induced HCC cells.8.RABIF promoted RAB10 expression.9.RABIF regulated lipophagy through RAB10.10.RABIF promoted proliferation,invasion and migration through RAB10.Conclusion:It was found in this study that RABIF promotes lipophagy and the cell proliferation,invasion,and migration ability of HCC cells through RAB 10,which might be a survival mechanism of tumor cells in response to metabolic stress,and provide a theoretical basis for the related mechanism of lipid catabolism in tumor metastasis.