Effect And Clinical Significance Of FOXA1 In Sorafenib Resistance In Hepatocellular Carcinoma

In recent years,with the continuous improvement of life quality,the incidence and mortality of hepatocellular carcinoma(HCC)have gradually increased,which has greatly affected people’s lives.In the first-line treatment of patients with advanced HCC,the multitarget tyrosine kinase inhibitor(TKI)sorafenib is the first first-line drug approved by the US Food and Drug Administration(FDA)for the treatment of advanced HCC.However,the problem of sorafenib resistance has become increasingly prominent in clinical practice,which seriously affects the survival benefit of patients with advanced HCC.At present,the specific molecular mechanism of sorafenib resistance is still unclear,and it is generally believed that tumor cells,tumor microenvironment and individual genetics are all involved.There is increasing evidence that further studies of sorafenib resistance can not only elucidate the specific molecular mechanisms,but also screen biomarkers that can be used for clinical monitoring,and contribute to the assessment of prognosis and the development of effective treatment strategies.To further explore the mechanisms of sorafenib resistance,we conducted the following studies:1.Screening and verification of key transcription factors related to epigenetic modification of sorafenib resistance in HCCFirstly,Sorafenib resistant cell lines were constructed by the method of low concentration increment combined with high dose intermittent shock.The sensitivity of the cells to Sorafenib,the expression of drug resistance genes and stemness related genes were also determined.RNA-seq and ChIP-seq technologies were combined to map the epigenetic modifications and transcriptome of sorafenib resistant cells in HCC.The epigenetic modification and transcriptome data of sorafenib resistance in HCC were then integrated.Motif analysis was performed on peaks with significantly enhanced H3K27ac signal in Sorafenib resistant cells in ChIP-seq.Combined with RNA-seq data,transcription factors with significantly enhanced promoter and enhancer H3K27ac signals and upregulated RNA levels in sorafenib-resistant cell lines were screened.Through further analysis of the public database,the transcription factor FOXA1 was finally screened.The mRNA and protein levels of FOXA1 in Sorafenib sensitive and resistant cells were verified by qRT-PCR and Western blot,respectively.The results showed that the expression level of FOXA1 in Sorafenib resistant cells was significantly up-regulated.2.The role of FOXA1 in the occurrence and development of HCCIn the above studies,FOXA1 was preliminarily identified to be significantly correlated with sorafenib resistance to HCC,but the role of FOXA1 in tumor,especially in the occurrence and development remains unclear in HCC.In order to reveal the function of FOXA1,FOXA1 knockdown sorafenib-resistant cell lines were constructed on the basis of the above experiments.CCK8 assay was used to detect the changes of sensitivity to sorafenib in resistant cell lines with stable knockdown of FOXA1.Cell scratch,Transwell and Matrigel assays were used to investigate the effect of FOXA1 on the invasion and migration of HCC cells.The effect of FOXA1 on the proliferation of HCC cells was investigated by ACEA and cloning assay.Our results showed that FOXA1 knockdown significantly increased the sensitivity of resistant cells to sorafenib and significantly decreased their proliferation,migration and invasion abilities.It is proved that FOXA1 plays an important role in regulating the occurrence,development and sorafenib resistance of HCC.3.Clinical significance of FOXA1 in hepatocellular carcinoma via public databaseTo explore the clinical significance of FOXA1 in HCC tissues,we used TCGA,UALCAN and GEO databases to analyze the expression level of FOXA1 in HCC tumor tissues.Survival analysis was used to verify the correlation between FOXA1 expression and the prognosis of HCC patients in the TCGA database.The role of FOXA1 in TME was also explained.Finally,a prognostic model based on FOXA1 expression was established and verified by ICGC and GEO database.Patients with high FOXA1 expression had worse survival.There were also significant differences in the characteristics of TME,with high expression of FOXA1 subtypes characterized by significant immunosuppression.Finally,a prognostic model based on FOXA1 expression and tumor staging was established,and verified by ICGC and GEO database.The model has certain predictive ability.In conclusion,ChIP-seq and RNA-seq have found that the expression of transcription factor FOXA1 is increased in sorafenib resistant cells,which may be involved in sorafenib resistance and metastasis in HCC,and provides an important basis for its clinical predictive value and molecular mechanism research.In addition,FOXA1 expression is significantly upregulated in clinical HCC tissues,and patients with high FOXA1 expression have worse survival and are characterized by significant immunosuppression.The prognostic model based on FOXA1 expression also has some predictive value.This study not only helps to reveal the molecular regulatory mechanism of FOXA1 in HCC development and sorafenib resistance,but also provides an effective novel molecular marker for the precise targeting of sorafenib.