| OBJECTIVE This study aims to explore the expression of long-chain acyl-CoA synthetases 4(ACSL4)in Hepatocellular carcinoma(HCC)and its associations with clinical pathological features and prognosis,and to investigate the role of ACSL4 on Huh7 cells in vitro.METHODS(1)By the use of immunohistochemistry staining in a tissue microarray consisting of 202 HCC tumor tissue and the adjacent normal tissues,the expression of ACSL4 protein was determined.The correlations between the protein expression of ACSL4 and clinical pathological features were analyzed by Chi-square test.The correlation between the protein expression of ACSL4 and prognosis was analyzed by Kaplan-Meier Log-rank test and Multivariate Cox regression analysis.(2)The effect of inhibiting ACSL4 activity by Rosiglitazone(a chemical inhibitor of ACSL4)on cell viability was determined in ACSL4-expressing Huh7 cells,especially under the condition of glucose deprivation.The cell viability was measured by flow cytometry.(3)Furthermore,the effect of silencing ACSL4 expression by specific ACSL4-siRNA(siRNA1-ACSL4 and siRNA2-ACSL4)and negative control siRNA on cell viability was also determined.(4)Western blotting was used to detect the expression of autophagy-related proteins,such as p-AMPK/total AMPK,p-S6/total S6,LC3II/I and P62.RESULTS(1)The immunohistochemical results suggested that(1)the proportions of positive protein expressions of ACSL4 were 61.9% and 34.8% in HCC and adjacent normal tissues respectively.The expression of ACSL4 protein in HCC tissue was significantly higher than that in adjacent normal tissue(p<0.001).Compared with adjacent normal tissues,56.3% of patients had higher expression of ACSL4 protein in HCC,and 13.3% of patients had lower expression of ACSL4 protein in HCC.30.4% of patients had no difference in the expression of ACSL4 protein between HCC and adjacent normal tissues.(2)In HCC tissue,the expression of ACSL4 protein was significantly correlated with the presence of diabetes mellitus(P=0.015),tumor necrosis(P=0.006),tumor capsule(P=0.044),serum AFP level(P=0.001)and histological grade(p=0.026),while not with other factors(p>0.05).(3)By the use of six years of follow-up data,the survival rate of the patients with positive expression of ACSL4 protein was significantly lower than those with negative ACSL4 expression(p=0.017).(4)Multivariate Cox regression analysis showed that the expression of ACSL4 protein,symptom,and recurrence within two years were the factors affecting the prognosis of HCC.(2)The results of cell viability showed that there was no significant difference in the viability rate between control and Rosiglitazone group when Huh7 cells were cultured with full medium(p>0.05).However,compared with the control group,the viability rate of Rosiglitazone group decreased by 55.73% under the condition of glucose deprivation(p<0.001).(3)There was no significant difference in the viability rate between negative control siRNA group and siRNA1-ACSL4,siRNA2-ACSL4 groups when Huh7 cells were cultured with full medium(p>0.05).However,compared with the negative control si RNA group,the viability rate of siRNA1-ACSL4 and siRNA2-ACSL4 groups decreased by 29.43% and 25.01% respectively under the condition of glucose deprivation(p<0.001).(4)The expressions of p-AMPK/total AMPK and LC3?/?protein were up-regulated(P<0.05)while p-S6/total S6 and P62 protein were down-regulated(P<0.05)under the condition of glucose deprivation.However,knocking down ACSL4 with specific siRNA did not effect the expression of p-AMPK/total AMPK,p-S6/total S6,LC3?/?and P62 protein(p>0.05).CONCLUSION(1)The expression of ACSL4 protein was significantly up-regulated in HCC,and was negatively correlated with tumor capsule and HCC prognosis.(2)ACSL4 inhibition and glucose deprivation could synergistically promote HCC cell death in vitro,which is consistent with the negative correlation between ACSL4 expression and tumor necrosis found in clinical tumor samples.(3)The site of ACSL4 may be located downstream of autophagy. |
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