FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA dictates human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA targeting of FOXA1 and FOXA2 in human hepatic (HepG2) cells and during hepatic differentiation significantly downregulated albumin (p < 0.05) and GRN TF gene expression (HNF4A, HEX, HNF1B, TBX3) (p < 0.05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid (GSC), FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < 0.05), all consistent with a partial/transient cell reprogramming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, and accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During hPSC-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate complex effects of FOXA1/2 on hepatic GRN effecting de-differentiation and metabolism with applications in studies of cancer, differentiation, and organogenesis.

中文翻译：

---

FOXA1/2 耗竭驱动人肝细胞系分化状态和代谢的全局重编程，并抑制人干细胞衍生的肝祖细胞分化

FOXA 因子是发育基因调控网络 (GRN) 的关键成员，该网络由主转录因子 (TF) 组成，可调节肠道和肝脏中的小鼠细胞命运和代谢。FOXA 如何决定人类肝细胞的命运、分化并同时调节代谢途径尚不清楚。在这里，我们旨在确定 FOXA2（和被认为可以补偿 FOXA2 的 FOXA1）在人肝细胞系 (HepG2) 中的肝分化和细胞代谢中的作用。siRNA 靶向人肝 (HepG2) 细胞和肝分化过程中的 FOXA1 和 FOXA2 显着下调白蛋白 (p < 0.05) 和 GRN TF 基因表达 (HNF4A、HEX、HNF1B、TBX3) (p < 0.05) 并显着上调内胚层/肠道/肝内胚层标志物（goosecoid (GSC)、FOXA3 和 GATA4)、肠道 TF (CDX2)、多能 TF (NANOG) 和神经外胚层 TF (PAX6) (p < 0.05)，都与部分/瞬时细胞重编程一致。shFOXA1/2 靶向导致了类似的发现并证明了可逆性的证据。RNA-seq 以及 shFOXA1/2 敲低 HepG2 细胞的生物信息学分析显示 235 个显着下调基因和 448 个上调基因，包括交替胚层谱系（心脏、内皮、肌肉）和神经外胚层（眼、神经）的标记上调。我们发现糖酵解、柠檬酸循环、线粒体基因的广泛下调，以及脂质代谢、磷酸戊糖途径和生酮的改变。功能代谢分析与这些发现一致，表明糖酵解和线粒体呼吸显着减少，以及脂滴的积累。我们假设 FOXA1/2 在体外抑制人类肝脏分化的开始。在 hPSC 肝分化过程中，siRNA 敲低显示去分化，并且出乎意料地激活了多能性因子和神经外胚层。shRNA 敲低显示了类似的结果和 SOX9（肝胆）的激活。这些结果证明了 FOXA1/2 对肝脏 GRN 的复杂影响，从而影响去分化和代谢，并在癌症、分化和器官发生的研究中得到应用。