DISSECTING MIRNA-MRNA REGULATORY NETWORK ALTERATIONS ASSOCIATED WITH RFX6 LOSS DURING HEPATIC DIFFERENTIATION

Abstract

RFX6 is a transcription factor with established roles in pancreatic islet development, yet its function during hepatic lineage progression remains largely unexplored. Given the shared endodermal origin of the liver and pancreas and the analogous temporal expression of RFX6 during hepatic progenitor specification, this study investigated whether RFX6 coordinates a miR–mRNA co-regulatory network during human hepatic differentiation and how its loss perturbs this network. Using CRISPR/Cas9-derived biallelic RFX6 knockout (KO) human induced pluripotent stem cell (iPSC) lines and their isogenic wild-type controls, cells were differentiated through sequential hepatic stages — definitive endoderm, posterior foregut, hepatic progenitors, and mature hepatocytes — using an established protocol. RFX6 expression was confirmed to peak at the hepatic progenitor stage and decline progressively upon maturation. miR sequencing revealed extensive miR dysregulation in RFX6 KO cells at both the hepatic progenitor stage (118 upregulated and 136 downregulated DEmiRs) and the mature hepatocyte stage (64 upregulated and 56 downregulated DEmiRs), with selected DEmiRs validated by qRT-PCR. Integrative miR–mRNA target analysis using IPA software identified stage-specific regulatory networks in which dysregulated miRs were predicted to target key hepatic genes governing lipid metabolism, bile acid synthesis, coagulation, glucose homeostasis, and extracellular matrix organization. Notably, critical transcriptional regulators of hepatic identity, including ONECUT2, FOXA1, and GATA4, showed persistent downregulation across both stages, suggesting sustained miR-mediated repression following RFX6 loss. Collectively, these findings establish that RFX6 plays an important role in maintaining stage-specific miR regulatory programmes during human hepatic differentiation, and that its loss disrupts a conserved post-transcriptional network with broad consequences for hepatic lineage commitment and functional maturation.

Date of Award	2026
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences