The Mechanism Of FOLR1 Enhancing Sorafenib Resistance In Hepatocellular Carcinoma By Regulating Folate Uptake

Objective: The death rate of hepatocellular carcinoma(HCC)ranks the third among all tumor-related deaths at home and abroad,accounting for about 85-90% of all primary liver malignancies.It is estimated that the number of cases of liver cancer will exceed 1 million by 2025.And more than half of HCC patients were diagnosed as advanced and had poor prognosis.For advanced patients,surgery and other treatment methods can no longer be the best choice for patients.Sorafenib,as a multi-target tyrosine kinase inhibitor,is applied to the treatment of advanced liver cancer.Clinical treatment data show that sorafenib can prolong the survival of patients with HCC.However,some patients usually develop clinical drug resistance to sorafenib within 6months of medication,thus reducing the therapeutic effect of sorafenib.Therefore,a comprehensive exploration of the molecular mechanism of sorafenib resistance in HCC is a crucial way to improve the clinical efficacy of sorafenib.In the early stage,our research team used proteomics to study the differentially expressed proteins of HCC Sorafenib resistant cell Huh7-R and Sorafenib sensitive cell Huh7,and found that folate receptor alpha(FOLR1),the key protein of drug resistance,was significantly overexpressed in Huh7-R.Further research found that FOLR1 overexpression promoted cell autophagy,thus causing Sorafenib resistance.However,the mechanism of autophagy caused by high expression of FOLR1 has not been clarified.Therefore,on this basis,this paper uses Western blot,fluorescence confocal microscopy,mass spectrometry and other techniques to explore the function and mechanism of FOLR1 resistance to sorafenib in HCC in vitro and in vivo experiments.Methods: 1.Detect the difference of folate uptake ability of Huh7-R and Huh7 groups,Huh7-NC1 and Huh7-FOLR1 groups,Huh7-R-NC and Huh7-R-SI groups.First,the three groups of cells were cultured for 24 hours in the medium containing folic acid(CY5-FA)with CY5 fluorescent label.The difference of fluorescence intensity was detected by fluorescence confocal microscope and flow cytometry,and the difference of FOLR1 expression in the three groups of cells was detected by Western blot;2.Detect the difference in the regulation of folate on autophagy level of three groups of cells.The REP-GFP-LC3 plasmid was transfected into cells with Lipofectamine LTX and Plus kit,and cultured in a medium containing 0 mg/L and 4mg/L folic acid.After fixation,the cells transfected with the plasmid were imaged with a fluorescence confocal microscope to detect the autophagy state of the cells.The expression level of autophagic marker protein LC3 B in the three groups of cells was detected by Western blot in the culture medium of 0 mg/L and 4 mg/L folic acid,respectively;3.Detect the difference of sensitivity of three groups of cells to sorafenib in the culture medium of 0 mg/L and 4 mg/L folic acid.CCK-8 and JC-1kits were used to detect the cell survival rate and apoptosis of three groups of cells in0 mg/L and 4 mg/L folic acid medium respectively;4.The effect of FOLR1 and folic acid on the therapeutic effect of sorafenib was verified by the tumor-bearing experiment in mice;5.Western blot was used to detect the phosphorylation level of ERK signal pathway and the expression level of autophagy marker protein LC3 B in Huh7 and Huh7-R groups,Huh7-NC1 and Huh7-FOLR1 groups in folic acid medium of 0 mg/L and 4 mg/L respectively;6.The mechanism of folic acid in the drug resistance of HCC to sorafenib was analyzed by quantitative proteomics without standard.Results: 1.FOLR1 is highly expressed in Huh7-R resistant cells and has stronger folate uptake ability compared with Huh7 cells.The expression of FOLR1 in Huh7-FOLR1 cells was higher than that of Huh7-NC1 in control group,and also showed stronger folate uptake ability.The expression of FOLR1 in Huh7-R-SI cells obtained by knockdown FOLR1 in Huh7-R-resistant cells was lower than that in Huh7-R-NC cells,and the uptake of folate was decreased;2.Huh7-R resistant cells showed higher level of autophagy in the medium with folate content of 4 mg/L.Huh7-FOLR1 cells showed strong autophagy in the medium with folate content of 4mg/L.After knockdown of FOLR1 of Huh7-R drug-resistant cells,the high level autophagy of cells in the culture medium with folate concentration of 4 mg/L decreased;3.Huh7-R resistant cells showed resistance to sorafenib in the medium with folate content of 4 mg/L,increased cell survival rate and decreased cell apoptosis.Huh7-FOLR1 cells with high expression of FOLR1 in Huh7 also showed resistance to sorafenib in the medium with folate content of 4 mg/L,increased survival rate and decreased apoptosis.After knockdown of FOLR1 of Huh7-R drug-resistant cells,the resistance of cells to sorafenib in the culture medium with folate concentration of 4mg/L decreased,the cell survival rate decreased,and the cell apoptosis increased;4.The tumor-bearing experiment in mice verified that Huh7-FOLR1 cells were tumor-bearing,and sorafenib treatment showed stronger sorafenib resistance under folate feeding conditions;5.The mechanism study found that the overexpression of FOLR1 activated the ERK signal pathway through folate uptake,thereby up-regulating autophagy;6.Further analyze the difference of proteomic expression of high expression FOLR1 after treatment with folic acid at 0 mg/L and 4 mg/L concentrations,and carry out KEGG enrichment analysis of the differential proteins.It is found that the protein processing,folic acid carbon pool,oxidative phosphorylation and other folate metabolism-related pathways in the endoplasmic reticulum are mainly concentrated,indicating that FOLR1 enhances cell autophagy by regulating these signal pathways.Conclusion: In this study,it was confirmed that the high expression of FOLR1 protein can enhance the ability of cells to absorb folic acid,and the high expression of FOLR1 in folic acid environment can enable Huh7 cells to obtain sorafenib resistance in vivo and in vitro.The molecular mechanism study found that the high expression of FOLR1 promoted folate uptake by activating ERK signal pathway,and regulating the protein processing,folate carbon pool,oxidative phosphorylation and other folate metabolism related pathways in the endoplasmic reticulum,so as to up-regulate the level of autophagy and enhance the drug resistance of liver cancer sorafenib.