Expression Of Foxa2 Research Articles

SND1 facilitates the invasion and migration of cervical cancer cells by Smurf1-mediated degradation of FOXA2

Staphylococcal nuclease domain-containing protein 1 (SND1) is known to be involved in the progression of a variety of human cancers. However, the role of SND1 in cervical cancer remains unclear. Here, we found that the expression of SND1 in cervical cancer tissue was higher than that in normal cervical tissue. Importantly, high SND1 expression was closely associated with tumorigenic phenotype and shorter survival among cervical cancer patients. Functional assays demonstrated that SND1 knockdown inhibited the migration and invasion capabilities of cervical cancer cells in vitro. Additionally, a xenograft assay showed that silencing SND1 in cervical cancer cells suppressed lung metastasis in vivo. Further investigation revealed that knockdown of SND1 inhibited epithelial-to-mesenchymal transition (EMT) of cervical cancer cells by enhancing FOXA2 expression. Moreover, the pro-metastasis effect of SND1 in cervical cancer was at least in part dependent on FOXA2 inhibition.Mechanistically, we found that SND1-induced FOXA2 ubiquitination resulted in degradation, mediated by the E3 ligase enzyme Smurf1. In summary, SND1 plays a crucial role in cervical cancer metastasis, and we provide evidence that SND1 may serve as a prognostic and therapeutic target in cervical cancer.

Molecular characterization of pulmonary defenses against bacterial invasion in allergic asthma: The role of Foxa2 in regulation of β-defensin 1.

Allergic asthma, characterized by chronic airway Th2-dominated inflammation, is associated with an increased risk of infection; however, the underlying mechanisms are unclear. Forkhead box protein A2 (Foxa2) plays a critical role in Th2 inflammation and is associated with pulmonary defenses. To determining the role of Foxa2 in Th2-dominated lung inflammation against the invading bacteria, we established a mouse OVA-sensitized model, an Escherichia coli lung invasion model, and mice with conditional deletion of Foxa2 in respiratory epithelial cells. The number of bacteria in the lung tissue was counted to assess clearance ability of lung. Lung inflammation and histopathology was evaluated using HE and PAS staining. It was found that OVA-sensitized mice had decreased E. coli clearance, reduced Foxa2 expression, and decreased DEFB1 secretion. Conditional deletion of Foxa2 in respiratory epithelial cells led to decreased clearance of E. coli and impaired secretion of DEFB1, similar to the OVA-induced allergic condition. The impaired secretion of DEFB1 may be responsible for the increased risk of infection in the Th2-dominated airway inflammation. Dual luciferase assay demonstrated that Foxa2 regulates DEFB1 expression by affecting its promoter activity in HBE cells. Our study indicated that Foxa2 plays an important role in Th2-dominated airway inflammation against invading bacteria. Conditional deletion of Foxa2 in respiratory epithelial cells can reduce pulmonary’s defense against bacterial invasion by inhibiting DEFB1expression.

MiR-130a-5p/Foxa2 axis modulates fetal lung development in congenital diaphragmatic hernia by activating the Shh/Gli1 signaling pathway

AimsCongenital diaphragmatic hernia (CDH) is a lethal birth defect characterized by congenital lung malformation, and the severity of pulmonary hypoplasia directly affects the prognosis of infants with CDH. Using a nitrofen-induced CDH rat model, we previously reported that Foxa2 expression was downregulated in CDH lungs by proteomics analysis. Here, we investigate the role of miR-130a-5p/Foxa2 axis in lung development of the nitrofen-induced CDH and evaluate its potential role in vivo prenatal therapy. Main methodsNitrofen was orally administrated on embryonic day (E) 8.5 to establish a rat CDH model, and fetal lungs were collected on E13.5, E15.5, E17.5, E19.5 and E21.5. The binding sites of miR-130a-5p on Foxa2 mRNA were identified using bioinformatics prediction software and were validated via luciferase assay. The expression levels of miR-130a-5p and Foxa2 were detected using qRT-PCR, ISH, IHC and western blotting. The role of miR-130a-5p/Foxa2 axis in CDH-associated lung development was investigated in ex vivo lung explants. Key findingsWe found that Foxa2 was downregulated in CDH lung tissues, and Foxa2 upregulating improved CDH branching morphogenesis in ex vivo lung explants. Meanwhile, we also showed that miR-130a-5p was significantly upregulated in CDH lungs and thus inversely correlated with Foxa2. Increasing miR-130a-5p abundance with mimics decreases Foxa2-driven Shh/Gli1 signaling and inhibits branching morphogenesis in ex vivo lung explants. SignificanceThis study was the first to show that the miR-130a-5p/Foxa2 axis played a crucial role in CDH-associated pulmonary hypoplasia. These findings may provide relevant insights into the prenatal diagnosis and prenatal therapy of CDH.

The key regulation of miR-124–3p during reprogramming of primary mouse hepatocytes into insulin-producing cells

Based on the action of small molecule compounds, the efficiency of differentiation of mouse primary hepatocytes into insulin-producing cells (IPCs) was improved by changing the expression of miR-124–2p. Hepatocytes were transfected with microRNA-124–3p (miR-124–3p) mimic or inhibitor, followed by a chemical-defined culture system for maturation of IPCs. Then, detect the expression of insulin-related genes and protein and insulin secretion of each stage during differentiation. The expression of Foxa2, PDX1, NeuroD, insulin1, and insulin2 in IPCs in the miR-124–3p inhibition expression group was significantly upregulated, while the results were opposite in the miR-124–3p overexpression group. The results of cell immunofluorescence and glucose stimulation in vitro of the miR-124–3p inhibition expression group showed that the expression of insulin, PDX1, and C-peptide was increased, and the differentiation efficiency was higher than those of the control group and overexpression group. The primary mouse hepatocytes were successfully reprogrammed into IPCs by small-molecule compounds. We found that miR-124–3p plays a negative regulatory role in the differentiation of hepatocytes into IPCs in vitro. Inhibition of miR-124–3p expression significantly increased the expression of FOXA2 and PDX1, promoted the differentiation of hepatocytes into IPCs, and increased the induction efficiency.

MiR-942 regulates the function of breast cancer cell by targeting FOXA2.

MicroRNA (MiR)-942 regulates the development of a variety of tumors, however, its function in breast cancer (BCa) has been less reported. Therefore, the present study investigated the regulatory effects of miR-942 on BCa cells. The expression of miR-942 in whole blood samples and BCa cell lines was detected by quantitative real-time (qRT)-PCR. Direct target gene for miR-942 was confirmed by dual-luciferase reporter assay. FOXA2 expression in adjacent tissues was detected by qRT-PCR. The effects of miR-942, or miR-942 with FOXA2, on the cell viability, proliferation, apoptosis, migration and invasion of BCa cells were determined by cell counting kit-8 (CCK-8), colony formation assay, flow cytometry, wound scratch and Transwell, respectively. The levels of N-Cadherin, E-Cadherin and Snail were determined by Western blot. Kaplan–Meier was used to explore the relationship among the expressions of miR-942 and FOXA2 and the prognosis of BCa patients. MiR-942 had high expressed in BCa, while its low expression significantly suppressed the cell viability, proliferation, migration and invasion of BCa, but increased cell apoptosis. Down-regulation of N-Cadherin and Snail and up-regulation of E-Cadherin were also induced by low-expression of miR-942. FOXA2, which was proved as the direct target gene for miR-942 and was low-expressed in BCa, partially reversed the effect of overexpressed miR-942 on promoting cell viability, proliferation, migration and invasion, and suppressed cell apoptosis. A lower survival rate was observed in BCa patients with a high expression of miR-942 and a low expression of FOXA2. MiR-942 promoted the progression of BCa by down-regulating the expression of FOXA2.

Loss of FoxA2 accelerates neoplastic changes in the intrahepatic bile duct partly via the MAPK signaling pathway.

Background: Intrahepatic cholangiocarcinoma (ICC) is characterized by a highly aggressive nature and a dismal outcome. FOXA2 is an archetypal transcription factor involved in cholangiocyte proliferation.Results: FOXA2 expression was negatively correlated with tumor stage (p = 0.024). Univariate and multivariate analyses showed that low FoxA2 expression was associated with tumor relapse and survival. At 20 weeks after TAA administration, FoxA2-/- mice displayed significant manifestations of neoplasia, while WT mice did not.RNA sequencing analysis showed that the expression of genes in the MAPK signaling pathway was significantly higher in FoxA2-/- mice. IHC and Western blot results showed that p-ERK1/2, CREB1 and RAS were highly expressed in FoxA2-/- mice. Furthermore, using in vitro experiments with siRNA, we found that low expression of FoxA2 could exacerbate the metastatic potential of ICC. The expression of p-ERK1/2 and RAS, which are key mediators of the MAPK signaling pathway, was significantly increased.Conclusion: Low FOXA2 expression negatively affected the prognosis of patients with ICC. Loss of FoxA2 expression could promote intrahepatic bile duct neoplasia partly via activation of the MAPK signaling pathway.Materials and methods: In all, the data of 85 patients with ICC were retrospectively collected and analyzed. TAA was used to induce ICC in FoxA2-/- mice and WT mice. RNA-sequencing analysis was used to identify the expression of different genes.

Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector

BackgroundHuman fibroblasts can be reprogrammed into induced hepatocyte-like cells through the expression of a set of transcription factors. Although the generation of induced hepatocyte-like cells by HNF4A, HNF1A, and FOXA3 expression has proven to be a robust experimental strategy, using multiple lentivirus results in a highly variable heterogeneous population.MethodsWe designed and implemented a novel approach based on the delivery of reprogramming factors and green fluorescent protein in a single doxycycline-inducible lentiviral vector using 2A self-cleaving peptides.ResultsFibroblasts infected with the lentiviral vector can be amplified in basic fibroblast culture media in the absence of doxycycline without induction of hepatic genes. Upon switching to hepatic maturation media containing doxycycline, cells stop proliferating, activate hepatic gene transcription, and perform metabolic functions characteristic of hepatocytes.ConclusionOur strategy can generate an unlimited source of homogeneously induced hepatocyte-like cells from different genetic background donors, capable of performing typical hepatic functions suitable for drug research and other in vitro applications.

Down-regulation of SOX17, GATA4 and FoxA2 promotes differentiation potential of hepatocytes from human hematopoietic stem cells

The failure of regeneration of damaged liver in various end-stage liver diseases results in high morbidity and mortality. In this context, we have demonstrated the differentiation ability of human hematopoietic stem cells (HSCs) into hepatocytes. In this study, HSCs were isolated from a donor and cultured which exhibited the presence of CD34 and CD133 and absence of CD90 and CD73 markers. These CD34+ HSCs were induced for 21 days in hepatocyte differentiation medium (HDM). The obtained cells were characterized by immunocytochemical, immunofluorescence, western blot, qRT-PCR and flow cytometry analysis. Further, functional assays were done to accentuate the differentiated cells are hepatocytes. In HDM at day 6 differentiated cells showed the expression of definitive endodermal (DE) markers, SOX17, GATA4 and FoxA2 indicating the beginning of differentiation process. At day 21 the flow cytometry analysis showed 84.2 % positive to ALB-PE, 75.4 % positive to HNF4α-PE, and 77.3% positive to AFP-PE. Further, the qRT-PCR and western blot analysis presented prominent expression of hepatocyte-specific genes AAT, ALB, AFP, CK18, CK19, HNF4α, TFR2, and Hepcidin confirms the generation of hepatocytes in HDM. The ability of albumin secretion, urea production, glycogen storage, uptake of LDL, high ALDH enzyme activity describes the functionality of differentiated hepatocytes. Distinct expression of UGT1A1, CYP2B6, CYP2C9, CYP3A4, and CYP7A1 genes explains the ability to clear toxins and bilirubin as observed in normal hepatocytes. All these results indicate HSCs were differentiated into hepatocytes thus, autologous transplantation of HSCs could be a better option in the regeneration of the damaged liver.

Down-regulation of aquaporin 9 gene transcription by 10-hydroxy-2-decenoic acid: A major fatty acid in royal jelly.

10‐Hydroxy‐trans‐2‐decenoic acid (10H2DA) is a unique lipid component of royal jelly produced by worker honeybees that exerts insulin‐like effects. We herein investigated the effects of 10H2DA on the gene expression of aquaporin 9 (AQP9), which functions as a glycerol transporter in the liver, to clarify whether 10H2DA modulates energy metabolism. 10H2DA suppressed AQP9 gene expression in HepG2 cells by promoting the phosphorylation of Akt and AMP‐activated protein kinase (AMPK). This suppression was partially recovered by the treatment of cells with inhibitors for Akt and AMPK. Based on the result showing that leptomycin B partially recovered the suppression of AQP9 gene expression, 10H2DA inhibited the expression of Foxa2, a transcription factor for the AQP9 gene, and also induced its nuclear exclusion. Although 10H2DA up‐regulated phosphoenolpyruvate carboxykinase and glucose‐6‐phosphatase gene expression, this was suppressed through the modulation of Foxa2 by insulin. These results suggest that 10H2DA suppresses AQP9 gene expression through the phosphorylation of Akt and AMPK and down‐regulation of Foxa2 expression.

2-Amino-4-(1-piperidine) pyridine exhibits inhibitory effect on colon cancer through suppression of FOXA2 expression.

The present study was aimed to investigate the effect of 2-amino-4-(1-piperidine) pyridine on migration and invasion of colon cancer cells. Treatment of colon cancer cells with 2-amino-4-(1-piperidine) pyridine reduced viability in concentration-based manner. The migration potential of HCT116 and HT29 cells was also suppressed on treatment with 2-amino-4-(1-piperidine) pyridine. In HCT116 and HT29 cells, apoptotic cell proportion was increased significantly by 2-amino-4-(1-piperidine) pyridine treatment. The expression of EMT and Vimentin in HCT116 and HT29 cells was reduced markedly on treatment with 2-amino-4-(1-piperidine) pyridine. The expression of E-cadherin was increased in HCT116 and HT29 cells by 2-amino-4-(1-piperidine) pyridine treatment. Treatment with 2-amino-4-(1-piperidine) pyridine reduced the expression of FOXA2 in HCT116 and HT29 cells. The 2-amino-4-(1-piperidine) pyridine treatment reduced growth of tumor in vivo in mice model. In summary, 2-amino-4-(1-piperidine) pyridine treatment inhibits colon cancer cell proliferation through down-regulation of FOXA2 expression. Therefore, 2-amino-4-(1-piperidine) pyridine can be used for the treatment of colon cancer.

H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling.

SummaryCellular responses to transforming growth factor β (TGF-β) depend on cell context. Here, we explored how TGF-β/nodal signaling crosstalks with the epigenome to promote mesendodermal differentiation. We find that expression of a group of mesendodermal genes depends on both TRIM33 and nodal signaling in embryoid bodies (EBs) but not in embryonic stem cells (ESCs). Only in EBs, TRIM33 binds these genes in the presence of expanded H3K18ac marks. Furthermore, the H3K18ac landscape at mesendodermal genes promotes TRIM33 recruitment. We reveal that HDAC1 binds to active gene promoters and interferes with TRIM33 recruitment to mesendodermal gene promoters. However, the TRIM33-interacting protein p300 deposits H3K18ac and further enhances TRIM33 recruitment. ATAC-seq data demonstrate that TRIM33 primes mesendodermal genes for activation by maintaining chromatin accessibility at their regulatory regions. Altogether, our study suggests that HDAC1 and p300 are key factors linking the epigenome through TRIM33 to the cell context-dependent nodal response during mesendodermal differentiation.

Connexin 43 Functions as a Positive Regulator of Stem Cell Differentiation into Definitive Endoderm and Pancreatic Progenitors.

SummaryEfficient stem cell differentiation into pancreatic islet cells is of critical importance for the development of cell replacement therapies for diabetes. Here, we identify the expression pattern of connexin 43 (Cx43), a gap junction (GJ) channel protein, in human embryonic stem cell (hESC)-derived definitive endoderm (DE) and primitive gut tube cells, representing early lineages for posterior foregut (PF), pancreatic progenitors (PP), pancreatic endocrine progenitors (PE), and islet cells. As the function of GJ channels is dependent on their gating status, we tested the impact of supplementing hESC-derived PP cell cultures with AAP10, a peptide that promotes Cx43 GJ channel opening. We found that this treatment promotes the expression of DE markers FoxA2 and Sox17, leads to a more efficient derivation of DE, and improves the yield of PF, PP, and PE cells. These results demonstrate a functional involvement of GJ channels in the differentiation of embryonic stem cells into pancreatic cell lineages.

The Club Cell Marker SCGB1A1 Downstream of FOXA2 is Reduced in Asthma.

Human SCGB1A1 protein has been shown to be significantly reduced in BAL, sputum, and serum from humans with asthma as compared with healthy individuals. However, the mechanism of this reduction and its functional impact have not been entirely elucidated. By mining online datasets, we found that the mRNA of SCGB1A1 was significantly repressed in brushed human airway epithelial cells from individuals with asthma, and this repression appeared to be associated with reduced expression of FOXA2. Consistently, both Scgb1A1 and FoxA2 were downregulated in an ovalbumin-induced mouse model of asthma. Furthermore, compared with wild-type mice, Scgb1a1 knockout mice had increased airway hyperreactivity and inflammation when they were exposed to ovalbumin, confirming the antiinflammatory role of Scgb1a1 in protection against asthma phenotypes. To search for potential asthma-related stimuli of SCGB1A1 repression, we tested T-helper cell type 2 cytokines. Both IL-4 and IL-13 repressed epithelial expression of SCGB1A1 and FOXA2. Importantly, infection of epithelial cells with human rhinovirus similarly reduced expression of these two genes, which suggests that FOXA2 may be the common regulator of SCGB1A1. To establish the causal role of reduced FOXA2 in SCGB1A1 repression, we demonstrated that FOXA2 was required for SCGB1A1 expression at baseline. FOXA2 overexpression was sufficient to drive promoter activity and expression of SCGB1A1 and was also able to restore the repressed SCGB1A1 expression in IL-13-treated or rhinovirus-infected cells. Taken together, these findings suggest that low levels of epithelial SCGB1A1 in asthma are caused by reduced FOXA2 expression.

Opposing Roles of the Forkhead Box Factors FoxM1 and FoxA2 in Liver Cancer

The forkhead box transcription factor FoxM1 is essential for hepatocellular carcinoma (HCC) development, and its overexpression coincides with poor prognosis. Here, we show that the mechanisms by which FoxM1 drives HCC progression involve overcoming the inhibitory effects of the liver differentiation gene FoxA2. First, the expression patterns of FoxM1 and FoxA2 in human HCC are opposite. We show that FoxM1 represses expression of FoxA2 in G1 phase. Repression of FoxA2 in G1 phase is important, as it is capable of inhibiting expression of the pluripotency genes that are expressed mainly in S-G2 phases. Using a transgenic mouse model for oncogenic Ras-driven HCC, we provide genetic evidence for a repression of FoxA2 by FoxM1. Conversely, FoxA2 inhibits expression of FoxM1 and inhibits FoxM1-induced tumorigenicity. Also, FoxA2 inhibits Ras-induced HCC progression that involves FoxM1. IMPLICATIONS: The observations provide strong genetic evidence for an opposing role of FoxM1 and FoxA2 in HCC progression. Moreover, FoxM1 drives high-grade HCC progression partly by inhibiting the hepatocyte differentiation gene FoxA2.

Sodium butyrate suppresses NOD1-mediated inflammatory molecules expressed in bovine hepatocytes during iE-DAP and LPS treatment.

Nucleotide oligomerization domain protein-1 (NOD1), a cytosolic pattern recognition receptor for the γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) is associated with the inflammatory diseases. Very little is known how bovine hepatocytes respond to specific ligands of NOD1 and sodium butyrate (SB). Therefore, the aim of our study was to investigate the role of bovine hepatocytes in NOD1-mediated inflammation during iE-DAP or LPS treatment or SB pretreatment. To achieve this aim, hepatocytes separated from cows at ∼160 days in milk (DIM) were divided into six groups: The nontreated control group (CON), the iE-DAP-treated group (DAP), the lipopolysaccharide-treated group (LPS), iE-DAP with SB group (DSB), LPS with SB group (LSB), and the SB group. Both iE-DAP and LPS highly increased the expression of both NOD1 and RIPK2, the two key factors for the immune response in hepatocytes. IκBα, NF-κB/p65, and MAP kinases (ERK, JNK, and p38) were activated through phosphorylation. The activation of NF-κB and MAPK pathway consequently increased the proinflammatory cytokines, IL-6, TNF-α, IL-8, and IFN-γ and the chemokines CCL5, CCL20, and CXCL-10. Both treatments improved iNOS/NOS2 expression. However, iE-DAP was failed to express acute phase protein SAA3, but HP and LPS HP but SAA3. These ligands also increased LRRK2, TAK1, TAB1, and β-defensins expression. The SB pretreatment at lower dose restored the function of hepatocytes by suppressing these increased molecules, as HDAC3 was inhibited. The activated NOD1 negatively regulated the expression of FOXA2. Altogether these data suggest an important role of bovine hepatocytes to promote immune responses via NOD1 expression during infection in the liver and a key role of SB to attenuate inflammation.

The long noncoding RNA Falcor regulates Foxa2 expression to maintain lung epithelial homeostasis and promote regeneration.

Transcription factors (TFs) are dosage-sensitive master regulators of gene expression, with haploinsufficiency frequently leading to life-threatening disease. Numerous mechanisms have evolved to tightly regulate the expression and activity of TFs at the transcriptional, translational, and posttranslational levels. A subset of long noncoding RNAs (lncRNAs) is spatially correlated with transcription factors in the genome, but the regulatory relationship between these lncRNAs and their neighboring TFs is unclear. We identified a regulatory feedback loop between the TF Foxa2 and a downstream lncRNA, Falcor (Foxa2-adjacent long noncoding RNA). Foxa2 directly represses Falcor expression by binding to its promoter, while Falcor functions in cis to positively regulate the expression of Foxa2. In the lung, loss of Falcor is sufficient to lead to chronic inflammatory changes and defective repair after airway epithelial injury. Moreover, disruption of the Falcor-Foxa2 regulatory feedback loop leads to altered cell adhesion and migration, in turn resulting in chronic peribronchial airway inflammation and goblet cell metaplasia. These data reveal that the lncRNA Falcor functions within a regulatory feedback loop to fine-tune the expression of Foxa2, maintain airway epithelial homeostasis, and promote regeneration.

Upregulated forkhead-box A3 elevates the expression of forkhead-box A1 and forkhead-box A2 to promote metastasis in esophageal cancer.

Esophageal cancer (EC) is one of the most lethal cancers currently known. Members of the forkhead-box A (FOXA) family, including FOXA1 and FOXA2, have been reported to regulate EC progression. However, the role of FOXA3, which is another FOXA member, has not yet been investigated. In the present study, public dataset analyses and immunohistochemistry of 96 samples from patients with EC were performed to determine the potential roles of FOXA3 in EC. The results revealed that FOXA3 was significantly upregulated in EC tumor tissues and Barrett's esophagus tissues. In addition, FOXA3 upregulation was positively associated with tumor invasion, distant metastasis, tumor-node-metastasis stage and shorter overall survival in patients with EC, and multivariate analysis identified FOXA3 as an independent prognostic marker. In vitro experiments demonstrated that the migratory and invasive abilities of EC109 and EC9706 cell lines were inhibited following FOXA3 knockdown. Notably, FOXA3 expression levels were positively correlated with FOXA1 and FOXA2 expression levels according to The Cancer Genome Atlas dataset analysis. Furthermore, FOXA3 knockdown decreased the expression levels of FOXA1 and FOXA2 in EC109 and EC9706 cell lines. Conversely, FOXA1 or FOXA2 overexpression compensated for the effects of FOXA3 knockdown on the migratory and invasive capacities of EC cells. In conclusion, the present study demonstrated that FOXA3 upregulation in EC cells promoted metastasis through regulation of other FOXA members.

Embryonic exposure to oxy-polycyclic aromatic hydrocarbon interfere with pancreatic β-cell development in zebrafish via altering DNA methylation and gene expression

Oxygenated polycyclic aromatic hydrocarbons (OPAHs) are a class of anthropogenic, persistent and very toxic PAH contaminant associated with developmental toxicity. Abnormal glucose metabolism disturbs energy balances that impair the early development of vertebrates, but the mechanisms by which maternal OPAH exposure alters glucose homeostasis in offspring are not well understood. Studies have suggested that epigenetic changes, particularly in DNA methylation, provide a memory of plastic developmental responses to the environment, leading to the generation of novel offspring phenotypes. The objective of this study is to test the hypothesis that embryonic exposure to low-dose OPAH can impair early β-cell differentiation in zebrafish (Danio rerio) by altering DNA methylation and gene expression. The zebrafish embryos were exposed to 0, 0.03, 0.1, 0.3, 1 and 3 μM 9‑fluorenone (9-FLO) at 3 h postfertilization (hpf) until 120 hpf to assess pancreatic organogenesis. 9-FLO exposure reduced total body length, eye length and heart rate, decreased insulin generation, interfered with glucose metabolism, and altered the expression of pancreatic organogenesis-related genes pdx-1, foxa2, isl1 and ptf1a. In particular, low-dose embryonic 9-FLO exposure significantly decreased β-cell differentiation marker gene pdx-1 mRNA levels, indicating that pancreatic endocrine is a more sensitive target response to embryonic low-dose OPAH exposure. Additionally, we found that DNA methyltransferases dnmt1 and dnmt3 were elevated and the DNA methylation at promoter regions of pdx-1 was increased at an early stage of development. These data demonstrated that the low-dose OPAH embryonic exposure can impair pancreatic endocrine development by increasing DNA methylation at the promoter regions of pdx-1 that are essential for β-cell differentiation.

Long Noncoding RNA LL35/Falcor Regulates Expression of Transcription Factor Foxa2 in Hepatocytes in Normal and Fibrotic Mouse Liver.

Long noncoding RNAs (lncRNA) play important roles in the regulation of transcription, splicing, translation, and other processes in the cell. Human and mouse lncRNA (DEANR1 and LL35/Falcor, respectively) located in the genomic environment in close proximity to the Foxa2 transcription factor were discovered earlier. In this work, tissue-specific expression of LL35/Falcor lncRNA has been shown in mouse liver and lungs. The use of antisense oligonucleotides allowed us to achieve LL35/Falcor lncRNA downregulation by 90%. As a result, the level of Foxa2 mRNA and protein dropped, which confirms the involvement of LL35/Falcor lncRNA in the regulation of transcription factor Foxa2. We have shown a decrease in the expression of LL35 lncRNA in liver fibrosis, which correlates with the previously published data for mRNA Foxa2. Thus, lncRNA LL35 regulates Foxa2 expression in the liver not only in normal conditions, but also during development of fibrosis, which allows one to consider lncRNA a biomarker of this pathological process.

Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells.

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317.