Establishment Of Hepatocellular Carcinoma 5-fluorouracil Resistant Cell Lines And Preliminary Research Of Its Drug Resistance Mechanism

Hepatocellular carcinoma(HCC)is a worldwide malignancy and has a yearly increased incidence with the changes of environment and lifestyle.Chemotherapy is an effective option for treating advanced HCC patients.5-Fluorouracil(5-FU),one of first-line chemotherapy drugs,often used in clinical treatment for advanced liver cancer.However,patients who treated by chemotherapy drugs for a long time can easily get the chemo-resistance to these drugs,resulting in a reduced or even ineffective treatment.At present,the mechanism of 5-FU resistance in HCC remains poorly understood.The aim of this study was to screen out critical genes and signaling pathways associated with the mechanism of 5-FU resistance.5-FU-resistant cells were established using stepwise increasing concentrations of 5-FU on parental HepG2 cells.Two kinds of single cell-derived clones(HepG2 and HepG2/5-FU)were obtained by a limited dilution method.Transcriptome sequencing(RNA-Seq)was performed to obtain the information of transcriptome between HepG2 and HepG2/5-FU.A total of 21409 genes were detected and then we analyzed the levels of gene expression and selected 641 differentially expressed genes(DEGs),of which 350 were up-regulated and 291 were down-regulated.After functional annotation and enrichment analysis of theses DEGs,we found that a total of 13 DEGs were enriched in the Wnt signaling pathway,and three of them had upstream and downstream relationships and their differences in expression were corresponded with expectations.They were the dickkopf Wnt signaling pathway inhibitor 1(DKK1),frizzled class receptor 10(FZD10),and dishevelled segment polarity protein 1(DVL1).Subsequently,real-time quantitative PCR(q-PCR)was used to validate the reliability of the RNA-Seq results and the differential expression of three Wnt signals(DDKI,FZD10 and DVL1).Finally,siRNA interference was performed to verificated the relevance between these three Wnt signals and 5-FU resistance in HepG2 cells.We screened out three new biomarkers(DKK1,FZD10 and DVL1)(located in the Wnt signaling pathway),whose regulation was associated with 5-FU resistance in hepatocellular cells.This study revealed that Wnt signaling pathway may play an important role in the formation of 5-FU resistance in hepatocellular carcinoma,which provides a potential target for individualized treatment of HCC patients.