Abstract

INTRODUCTION Peptidyl arginine deiminase 4 (PAD4) catalyzes the deamination of arginine residues to citrulline, a post-translational modification known as citrullination, which regulates protein structure, function, and localization. Despite growing evidence connects PAD4 to cancer progression, its role in intrahepatic cholangiocarcinoma (ICC) remains largely unexplored. OBJECTIVES This study aimed to elucidate the role and underlying mechanisms of PAD4 in the regulation of intrahepatic cholangiocarcinoma proliferation, as well as to explore its clinical relevance and therapeutic potential. METHODS Hydrodynamic tail vein injection (HTVI) and subcutaneous xenograft models were used to evaluate the role of PAD4 in vivo. In vitro functional assays were performed to assess the effects of PAD4 on ICC cell proliferation. Putative targets were identified by RNA-seq and Co-IP/MS analyses. Molecular interactions were examined using GST pull-down, surface plasmon resonance (SPR), and RNA immunoprecipitation-PCR. The clinical relevance of PAD4 was evaluated in ICC patient samples using immunohistochemical analysis. RESULTS PAD4 was identified as a key promoter of ICC proliferation by enhancing post-transcriptional expression of the MCM complex. Mechanistically, PAD catalytic domain of PAD4 interacted with the KH1 domain of IGF2BP2 and catalyzed its citrullination at R597, which markedly increased the affinity of IGF2BP2 for m6A-modified MCM2-7 transcripts, thereby stabilizing and elevating MCM mRNAs expression. Clinically, high PAD4 expression correlated with poor prognosis in ICC patients, while PAD4 and IGF2BP2 co-expression predicted worse outcomes. In vivo, combined inhibition of PAD4 and IGF2BP2 synergistically suppressed ICC growth. CONCLUSION