A Study On The Mechanism Of FABP5 Regulating 5-FU Drug Resistance In Human HCC

Objective: liver cancer is a kind of cancer with high malignant degree,and its main cause is hepatitis B virus(HBV)infection.As a large country of hepatitis B,there are a large number of new cases of liver cancer every year.More than 90% of the cases ’ Pathological type is hepatocellular carcinoma(HCC).At present,drug treatment accounts for a large proportion of all treatments to patients,but the emergence of drug resistance makes the improvement of overall survival rate not obvious.In HCC cell lines Bel7402 and Bel7402 resistant to 5-FU,we found that the expression of FABP5 in drug-resistant cell line Bel/5-FU was significantly higher than that in Bel7402 by mass spectrometry proteomic analysis.In view of the fact that the increase of FABP5 indicates adverse consequences in various cancers,we explored the role of FABP5 in the drug resistance of HCC to 5-FU and preliminarily explored the mechanism of its effect.Methods: 1.We searched the difference of FABP5 expression between hepatocellular carcinoma and normal liver tissue through GEPIA database,and detected the effect of FABP5 expression on the overall survival curve of patients.2.The drug resistance of Bel/5-FU cell line was verified by plate colony formation test and IC50 curve determined by CCK-8,while Bel7402 cell line was sensitive to 5-FU.3.To verify the proteomic results,we detected the expression of FABP5 in m RNA and protein levels in the two cell lines by q PCR and Western Blot methods respectively.We also examined the expression of FABP5 in other HCC cell lines Huh7 compared with the former two kinds of cells.We also detected the changes of FABP5 expression in Bel/5-FU cell lines cultured in different 5-FU drug concentrations.4.The construction of knock-down and over-expressed FABP5 cell lines were detected by q PCR and Western Blot experiments.5.Plate colony formation assay and CCK-8 were used to detect the effects of cell clone formation and cell proliferation after knockout and overexpression of FABP5.6.The expression of drug resistance-associated protein P-gp in the two cell lines was detected,and the effect of knock-down and overexpression of FABP5 on the expression of P-gp was detected.7.Search the database and consult the literature to find the downstream molecule PPARγ affected by FABP5,and Western Blot was used to detect the change of PPARγ expression level after knock-down and overexpression of FABP5.Result: 1.the results of GEPIA database showed that the expression of FABP5 in hepatocellular carcinoma tissue was significantly higher than that in normal liver tissue,and the overall survival rate of patients with high expression of FABP5 was significantly lower than that of patients with low expression,The difference is statistically significant(P<0.05).2.Plate colony formation assay and measuring IC-50 curve by CCK-8 assay showed that Bel/5-FU was resistant to 5-FU,while Bel7402 was sensitive to 5-FU.3.q PCR and Western Blot showed that the m RNA and protein levels of FABP5 in Bel/5-FU were significantly higher than those in Bel7402,The difference is statistically significant(P<0.05);and the protein expression of FABP5 in HCC cell line Huh7 was at a higher level;Bel/5-FU cell line cultured in different5-FU drug concentrations had no effect on FABP5 protein expression.4.Both q PCR and Western Blot showed that Bel/5-FU transient knockdown,Bel7402 stable over-expression and Bel/5-FU stable knockdown cell lines were successfully constructed,and the knockdown efficiency of si486 was the highest in the knock-down sequence.5.Overexpression of FABP5,in Bel7402 enhanced the tolerance of cells to 5-FU,while knocking down FABP5,in Bel/5-FU decreased the tolerance of cells to 5-FU.6.Molecular level Western Blot showed that the expression of drug resistance-related protein P-gp in Bel/5-FU was higher than that in Bel7402,and the expression of P-gp increased and decreased with the increase and decrease of FABP5 expression.7.FABP5 can positively regulate the expression of downstream nuclear transcription factor PPARγ.Conclusion: the increase of FABP5 is related to the poor prognosis of patients with HCC,suggesting that FABP5 may be a meaningful index and potential drug target in clinical diagnosis and treatment;the expression of FABP5 in drug-resistant cells of Bel/5-FU is increased,and knocking down FABP5 can reduce drug resistance,Overexpression of FABP5,in Bel7402 enhanced the drug resistance of cells to 5-FU;in HCC,FABP5 can regulate the expression of downstream nuclear transcription factor PPAR γ.