C/EBPα-ACS14 Promoting The Survival Of Hepatocellular Carcinoma Cells Under Metabolic Stress

Objective Hepatocellular carcinoma(HCC)is the most common primary tumor in the liver,with complex etiology and high mortality.The rapid growth of tumor and insufficient neoangiogenesis may cause tumors to face harsh metabolic stress such as hypoxia and glucose deficiency.Metabolic reprogramming is one of the hallmarks of cancer,in which fatty acid metabolism reprogramming can decompose the fat stored in cancer cells and provide the necessary energy for their survival.This project studies the influence of fatty acid metabolism-related enzyme ACSL4 on the survival of HCC cells under metabolic stress,and determines molecular mechanisms involved in how C/EBPa regulates ACSL4 expression.Indentificantion of the key sensitive metabolic pathways will help explore the mechanism of the occurrence and development of HCC and develop effective treatment methods.Methods(1)The survival rate of recurrent HCC patients were stratified by transarterial chemoembolization(TACE)treatment or not and analyzed based on the ACSL4 expression in primary HCC tissues.(2)Establishment of ACSLs family genes knockout cells:small interfering RNA(siRNA)was used to silence,and CRISPR/cas9 dual vector system to knock out ACSL3 or ACS14 genes in Huh7 cells.Their expressional changes were verified by Western blotting to validate silence and knockout effect.(3)Cell metabolism:We detected the changes of key energy metabolites of gene knockout cells under glucose starvation,including intracellular triglyceride,fatty acid beta oxidation product β-hydroxybutyrate,intracellular ATP.The oxygen consumption rate of mitochondria was measured by Seahorse mitochondrial pressure kit.(4)Cell death:Flow cytometry was used to detect the proportion of dead cells,the use of ACSL4 inhibitors(Triacsin C,Rosiglitazone),siRNA silencing and sgRNA knockout of ACSL3 or ACSL4 genes,and then synergized with glucose starvation.(5)Regulation of ACSL4 by C/EBPa:We first analyzed the correlation between the expression of ACSL4 and C/EBPa in HCC cell lines,and then detected the effect of knockout or overexpression of C/EBPα on ACSL4 gene expression.Bioinformatics methods were then applied to find the possible binding sites of C/EBPa on the ACSL4 5’-end promoter;dual-luciferase vector plasmids containing different length fragments of binding sites or mutations in the binding sites were then generated.The above-mentioned reporter vectors with Renilla vector were simultaneously transfected into control or C/EBPa knockout Huh7 cells,and the fluorescence intensity changes were detected.Results(1)Among patients undergoing TACE treatment in patients with recurrent HCC,the survival rate of patients with high expression of ACSL4 in tumor tissues was significantly lower than that of patients with low expression of ACSL4(P=0.003).In patients who were not treated with TACE,the expression of ACSL4 in tumor tissues was not associated with their survival rate(P=0.685).(2)The basic content of fatty acid beta oxidation product beta hydroxybutyric acid in ACSL3 and ACSL4 knockout cells did not change significantly.After 24 hours of glucose starvation,the beta hydroxybutyrate of the control cells and the ACSL3 knockout cells increased compared to the ACSL4 knockout cells.The basic triglyceride storage level of ACSL3 and ACSL4 gene knockout cells was lower than that of the control group.After 24 hours of combined glucose starvation,the triglyceride level of ACSL4 knockout cells increased.The basic ATP contents of ACSL3 and ACSL4 gene knockout cells were lower than that of control cells.After glucose starvation for 12 hours,the ATP levels of control cells and ACSL4 knockout cells further decreased.The results of the Seahorse energy metabolism experiment showed that ACSL4 knockout combined with glucose starvation caused a decrease in oxygen consumption rates related to ATP production,and the ACSL3 and ACSL4 knockout combined with glucose starvation caused a substantial increase of proton leak related to the loss of mitochondrial membrane permeability.(3)Inhibition of ACSL4 expression/activity through inhibitors,siRNA,Crispr/Cas 9-delivered sgRNA significantly promoted cell death induced by glucose starvation.However,these effects were absent in ACSL3-knockout cells.The ACSL4-mediated cell survival results in Hep3B cells with high expression of ACSL4 replicated those in Huh7 cells,but SNU-182 cells with low expression of ACSL4 cannot.(4)The results quantitative RT-PCR showed that C/EBPa mRNA was positively correlated with ACSL4 mRNA expression(R2=0.5793,P=0.047),but not with ACSL3 mRNA expression(P=0.409).The protein expression levels of ACSL3 and ACS 14 decreased with the knockout of C/EBPa,while the protein expression level of ACSL4 increased with the overexpression of C/EBPa.(5)Bioinformatics analysis predicted that there were multiple C/EBPa binding sites on the 5’promoter sequence of ACSL4,including-490 to-479,-1064 to-1053,-1335 to-1324 and-1612 to-1601.The results of the dual luciferase reporter assay found that the luciferase activity of the two vector plasmids containing ACSL4 promoter fragments-1066 to+1 and-1337 to+1 both increased in Huh7 cells,but not in the ACSL4-knockout cells.Mutation of the putatieve regulatory sequences-1064 to-1053 or-1335 to-1324 binding site alone,decreased luciferase activity.Double mutations of the two binding sites,significantly decreased luciferase activity.Conclusions(1)The survival rate of recurrent HCC patients with high expression of ACSL4 had poorer prognosis after TACE treatment.(2)ACSL4 knockout combined with glucose starvation caused cells cannot unable to utilize triglycerides for fatty acid beta oxidative decomposition energy of ATP.(3)ACSL4 protects HCC cells from cell death induced by glucose starvation.(4)C/EBP a has binding sites on the 5’-end promoter of ACSL4,so that it directly transcribes ACSL4.