Background:Our previous studies have shown that regulatory factor X5 (RFX5),a classical transcription regulator of MHCII genes,was obviously overexpressed in hepatocellular carcinoma (HCC) tumors.However,the role of RFX5 in the carcinogenesis and progress of HCC remains unknown.This study aimed to reveal its biological significance and the underlying mechanism in HCC.Methods:RFX5 mRNA expression level and copy number variation in HCC tumors and cell lines were determined by analyzing deposited data sets in the Cancer Genome Arlas and Gene Expression Omnibus database.The biological significance of RFX5 in HCC was investigated by monitoring the colony formation and subcutaneous tumor growth capacity when RFX5 was silenced with lentiviral short hairpin RNA and CRISPR/Cas9 system in HCC cell lines.The downstream gene transcriptionally activated by RFX5 in HCC cells was determined by chromatin immunoprecipitation and luciferase reporter assay.The involvement of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (YWHAQ) in HCC development was further determined by performing colony formation rescue assay and subcutaneous tumor growth rescue experiment.The association of YWHAQ with recurrence-free survival of patients with HCC was assessed by Kaplan-Meier analysis.Moreover,apoptosis level and the protein level of p53 pathway were determined to reveal the mechanism of RFX5 in driving HCC development.Results:RFX5 was amplified and highly overexpressed in HCC tumor tissues compared with the corresponding non-tumor tissues.The mRNA expression level of RFX5 was significantly correlated with its DNA copy number (r =0.4,P ＜ 0.001).Functional study demonstrated that RFX5 was required for both clonogenic forming in vitro and subcutaneous tumor growth in vivo of HCC cells.Further study identified YWHAQ,namely 14-3-3 tau,as a key downstream transcriptional target gene of RFX5,which was tightly regulated by RFX5 in HCC.Moreover,overexpression of YWHAQ largely rescued the clonogenic growth of HCC cells that was suppressed by RFX5 knockdown.In addition,overexpression of YWHAQ in primary tumor was linked to poor prognosis of patients with HCC.These results demonstrated that YWHAQ was a downstream effector of RFX5 in HCC.Notably,RFX5-YWHAQ pathway could protect cells from apoptosis by suppressing the p53 and Bax in HCC.Contusion:RFX5 is a putative HCC driver gene that plays an important role in the development and progression of HCC by transactivating YWHAQ and suppressing apoptosis.