Identification of cancer-related genes FGFR2 and CEBPB in choledochal cyst via RNA sequencing of patient-derived liver organoids.

BACKGROUND: Choledochal cysts (CC) are congenital bile duct anomalies with 6-30% risk for developing bile duct cancer. However, the molecular mechanisms underlying cancer risk of CC are unknown. We sought to identify the gene expression changes underlying the cancer risk of CC patients. METHODS: Liver organoids (n = 51) were generated from liver/bile duct biopsies of CC (n = 7; type I) and hepatoblastoma (n = 5; HB: non-tumor & tumor) for RNA sequencing. Bioinformatics analysis was conducted to identify differentially expressed cancer-related genes in CC and controls. We compared CC with non-cancerous and cancerous controls, normal adjacent non-tumor region of hepatoblastoma (HB) liver as non-cancerous control and tumor region as non-CC cancer control (HB-tumor). Reverse transcription real-time quantitative PCR (RT-qPCR) verification and immunohistochemistry of selected genes was conducted in additional CC and HB liver biopsies. FINDINGS: HB non-tumor and HB tumor organoids displayed distinct gene expression profiles. Expression profiling separated CC organoids into two clusters, one overlapping with HB non-tumor and the other one with HB tumor organoids. Genes selected based on their log2FoldChange values for RT-qPCR verification in 31 CC and 11 HB non-tumor liver tissues revealed significantly elevated expression of FGFR2 in 7 and CEBPB in 2 CC liver tissues (CC vs HB: 4.082 vs. 0.7671, p<0.01; 2.506 vs. 1.210, p<0.01). Distinctive positive staining in bile ducts were seen in CC, HB tumor and non-tumor liver tissues for FGFR2 and CEBPB. Percentages of CEBPB-immuno-positive or FGFR2-immuno-positive bile duct cells in CC and HB-tumor liver were higher than that in HB non-tumor liver. INTERPRETATION: The study identified dysregulated genes related to cancer pathways in CC patients suggesting cancer risk. The findings suggest that the elevated expression of FGFR2 and CEBPB in liver may contribute to cancer development in CC patients.

Developing Biliary Atresia-like Model by Treating Human Liver Organoids with Polyinosinic:Polycytidylic Acid (Poly (I:C)).

Background: We explored the feasibility of creating BA-like organoids by treating human liver organoids with Polyinosinic:Polycytidylic acid (Poly I:C). Methods: Organoids were developed from the liver parenchyma collected during Kasai portoenterostomy (BA) and surgery for other liver disorders (non-BA). The non-BA organoids were co-cultured with poly I:C (40 µg/mL). The organoid morphology from both samples was compared on day 17. RNA-sequencing was performed to examine the transcriptomic differences. Results: Non-BA liver organoids developed into well-expanded spherical organoids with a single-cell layer of epithelial cells and a single vacuole inside. After poly I:C treatment, the majority of these organoids developed into an aberrant morphology with a high index of similarity to BA organoids which are multi-vacuoled and/or unexpanded. RNA-sequencing analysis revealed that 19 inflammatory genes were commonly expressed in both groups. Conditional cluster analysis revealed several genes (SOCS6, SOCS6.1, ARAF, CAMK2G, GNA1C, ITGA2, PRKACA, PTEN) that are involved in immune-mediated signaling pathway had a distinct pattern of expression in the poly I:C treated organoids. This resembled the expression pattern in BA organoids (p < 0.05). Conclusions: Poly I:C treated human liver organoids exhibit morphology and genetic signature highly compatible to organoids developed from BA liver samples. They are potential research materials to study immune-mediated inflammation in BA.

Deletion of interleukin enhancer binding factor 2 (ILF2) resulted in defective biliary development and bile flow blockage.

PURPOSE: Biliary atresia (BA) is a devastating obstructive bile duct disease of newborns. BA has the highest incidence in Asians (1/5000), and its pathogenesis is unclear. We identified BA-private rare copy number variants (CNVs; 22 duplications and 6 deletions). ILF2 gene locates in the chromosome region (Chr1:153410347-153,634,058) which was deleted in a nonsyndromic BA patient. However, it is still not known whether ILF2 plays a role in hepatobiliary development and its deletion impacts on the bile duct development. METHODS: To investigate if ILF2 is required for biliary development, we knock-out the zebrafish homologs of ILF2 by CRISPR/Cas9 approach, and discover that deletion of ILF2 causes a defective biliary development and a lack of bile flow from the liver to the gall bladder in zebrafish, which is a resemblance of phenotypes of BA. RESULTS: Our data indicate that ILF2 gene is required for biliary development; deletion of ILF2 impairs bile duct development and could contribute to BA pathogenesis. This will be the first study to functionally evaluate the genes interfered by BA-private CNVs in hepatobiliary development and in BA pathogenesis. CONCLUSIONS: Such functional study may reveal the potential value of these BA-private CNVs in the disease pathogenesis for BA. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Beta-amyloid deposition around hepatic bile ducts is a novel pathobiological and diagnostic feature of biliary atresia.

BACKGROUND AND AIMS: Biliary atresia (BA) is a poorly understood and devastating obstructive bile duct disease of newborns. It is often diagnosed late, is incurable and frequently requires liver transplantation. In this study, we aimed to investigate the underlying pathogenesis and molecular signatures associated with BA. METHODS: We combined organoid and transcriptomic analysis to gain new insights into BA pathobiology using patient samples and a mouse model of BA. RESULTS: Liver organoids derived from patients with BA and a rhesus rotavirus A-infected mouse model of BA, exhibited aberrant morphology and disturbed apical-basal organization. Transcriptomic analysis of BA organoids revealed a shift from cholangiocyte to hepatocyte transcriptional signatures and altered beta-amyloid-related gene expression. Beta-amyloid accumulation was observed around the bile ducts in BA livers and exposure to beta-amyloid induced the aberrant morphology in control organoids. CONCLUSION: The novel observation that beta-amyloid accumulates around bile ducts in the livers of patients with BA has important pathobiological implications, as well as diagnostic potential. LAY SUMMARY: Biliary atresia is a poorly understood and devastating obstructive bile duct disease of newborns. It is often diagnosed late, is incurable and frequently requires liver transplantation. Using human and mouse 'liver mini-organs in the dish', we unexpectedly identified beta-amyloid deposition - the main pathological feature of Alzheimer's disease and cerebral amyloid angiopathy - around bile ducts in livers from patients with biliary atresia. This finding reveals a novel pathogenic mechanism that could have important diagnostic and therapeutic implications.

Conditional deletion of platelet derived growth factor receptor alpha (Pdgfra) in urorectal mesenchyme causes mesenchyme apoptosis and urorectal developmental anomalies in mice.

In mammals, urorectal development starts at early embryonic stage, defective urorectal development results in anorectal malformations, which are common congenital developmental defects of the anus and the urethra in newborns. The etiology and embryology of the defects are still largely unknown. Platelet-derived growth factor receptor alpha (Pdgfra) is a cell surface receptor tyrosine kinase, upon binding to its ligands (Pdgfa-d), mediates intracellular signaling and regulates embryonic development. The expression of Pdgfra is tightly regulated in the developing urorectal mesenchyme, and its dysregulation is associated with urorectal defects in animals with urorectal defects. Knockout of Pdgfra induces early embryo lethality which precludes investigation of Pdgfra in urorectal development. To address the temporal requirement of Pdgfra in urorectal development, we conditionally deleted Pdgfra in Pdgfra-expressing tissues using a tamoxifen inducible Cre-loxP approach in mice, examined the urorectal development in Pdgfra conditional knockout (Pdgfra-cKO) embryos. We showed that conditional deletion of Pdgfra in Pdgfra-expressing tissues at E10-E11 caused cloaca septation defect, anteriorly displaced anus, defective urogenital folds development and abnormal urethra tubularization in both male and female mice. Furthermore, we showed that Pdgfra was required for the survival of urorectal mesenchyme, deletion of Pdgfra caused apoptosis in the peri-cloacal, the peri-urethra and the urorectal septum mesenchyme of Pdgfra-cKO mutants, associated with an induction of p53, Ndrg1 and activation of caspase-3 in Pdgfra-cKO embryos. In conclusion, Pdgfra is required for the development and survival of the urorectal mesenchyme in embryo, dysregulated Pdgfra signaling induced urorectal defects in mice resembling human congenital diseases of anorectal malformations and hypospadias. Perturbation of PDGFRA signaling may contribute to anorectal malformations and hypospadias in human.

Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

BACKGROUND: Platelet-derived growth factor receptor alpha (PDGFRα) is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures. PURPOSE: To address the temporal requirement of Pdgfra in embryonic development. METHODS: We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies. RESULTS: Current study showed that (i) conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5) resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii) the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives. CONCLUSION: Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a) the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b) if mutations / sequence variations of these regulatory elements cause these anomalies.

Stimulating effects of dopamine on chloride transport across the rat caudal epididymal epithelium in culture.

The present study investigated the effects of dopamine on chloride transport across cultured rat caudal epididymal epithelium. The results showed that dopamine induced a biphasic short-circuit current (Isc) in a concentration-dependent manner. The dopamine-induced response consisted of an initial rapid spike followed by a sustained phase. The alpha and beta adrenoreceptor inhibitors, phentolamine and propranolol, inhibited the initial spike and the sustained phase, respectively, suggesting a contribution of adrenergic receptors. The response was almost abolished by removing the extracellular Cl-, suggesting that the dopamine-induced short-circuit current is primarily a Cl- current. The response was inhibited by the apical Cl- channel blocker, diphenylamine-dicarboxylic acid, and the Ca2+-activated Cl- channel blocker, disulfonic acid stilbene, indicating that Cl- may pass through two types of Cl- channels on the apical side. Preloading monolayers with the intracellular Ca2+ chelator BAPTA/AM abolished the initial spike and greatly reduced the second phase in the Isc response to dopamine. Pretreating the monolayers with an adenylate cyclase inhibitor, MDL12330A, inhibited all of the second Isc response and part of the initial spike. Also, characteristics of the Cl- currents induced by dopamine were observed in whole-cell patch-clamp recording. The increases of intracellular cAMP and Ca2+ induced by dopamine were also measured. The results suggest that extracellular dopamine activates Ca2+-dependent and cAMP-dependent regulatory pathways, leading to activation of both Ca2+-dependent and cAMP-dependent Cl- conductances in epididymal epithelial cells.

Innate immune responses of epididymal epithelial cells to Staphylococcus aureus infection.

The epithelium is an active participant in the host response to infection. We hypothesized that epididymal epithelia play a role in the innate immune responses by sensing the presence of pathogens, expressing and secreting inflammatory cytokines that recruit inflammatory cells in response to invading pathogens. Our results indicated that TNF-alpha and IL-1beta could be secreted by the primary cultured rat epididymal cauda epithelia infected with Staphylococcus aureus. Epididymal epithelial-induced nitric oxide synthase (iNOS) expression was up-regulated after S. aureus infection and nitric oxide (NO) was also found to be produced significantly. NF-kappaB inhibitor BAY11-7082 inhibited TNF-alpha secretion completely and p38 mitogen-activated protein kinases (MAPKs) inhibitor SB203580 decreased TNF-alpha secretion partly, indicating that NF-kappaB and p38 signal pathways were involved in this inflammation response. Toll-like receptor (TLR)-2 and -4 were shown to be expressed in primary cultured rat epididymal epithelia. After infection the level of TLR2 expression was up-regulated rather than TLR4. These results demonstrated that epididymal epithelium have an innate immune response through activation of p38 MAPK and NF-kappaB after TLR2 activation by S. aureus infection.

Cellular mechanism of adrenalin stimulated chloride secretion via beta-adrenoceptor in T84 cells.

In the present study, the intracellular regulatory pathways involved in the adrenalin-stimulated chloride secretion across T84 cells were investigated. Biphasic characteristics were observed in the Isc response to the basolateral addition of adrenalin (0.25 nM-100 microM). The biphasic response was almost abolished by removing ambient Cl(-). Chloride secretion was found to depend on the activities of basolaterally located Na+-K+-2Cl(-) cotransporters and K+ channels. The alpha-adrenoceptor antagonist phentolamine did not have any effect on either phase of adrenalin-induced Isc, while after pretreatment of the beta-adrenoceptor antagonist propranolol, the adrenalin-induced Isc was substantially abolished, suggesting the biphasic response may be mediated by the beta-adrenoceptor. Under whole cell patch-clamp conditions, T84 cells responded to adrenalin with a rise in inward current. The current, which exhibited a linear I-V relationship and time- and voltage-independent characteristics, was inhibited by the chloride channel blocker DPC and the reverse potential was close to the equilibrium potential for Cl(-) (0 mV), implying that the current was Cl(-) selective. When preloaded with a Ca2+-chelating agent, BAPTA/AM did not affect the Isc response to adrenalin, whereas the Isc was destroyed by pretreating the cells with an adenyl cyclase inhibitor, MDL12330A. These observations were further supported by the intracellular [cAMP] measurement experiment, indicating that adrenalin induced chloride secretion could be mediated by a beta-adrenoceptor only involving cAMP as an intracellular second messenger.

Clonorchis sinensis: molecular cloning and functional expression of a novel cytosolic glutathione transferase.

Glutathione transferases (GSTs) represent a large family of enzymes. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis (Cs), we isolate another cDNA clone encoding a novel cytosolic GST enzyme. To discriminate with our former reported CsGST, we designated this GST as CsGST1. This new cDNA contains 744 bp with a putative open reading frame of 213 amino acids. The deduced amino acid sequence exhibits 88% identity to Opisthorchis viverrini 28GST (Ov28GST), 60 and 52% identity to C. sinensis cytosolic 28-kDa GST (Cs28GST) and CsGST, respectively. The CsGST1 was expressed in Escherichia coli BL21(DE3) as a His-tag fusion protein and was purified by Ni-NTA agarose. The recombinant CsGST1 showed moderate GST activity of 0.79 U mg(-1). The average K (m) of the CsGST1 for 1-chloro-2, 4-dinitrobenzene is 150 microM. Cibacron blue F3GA and albendazole inhibited the CsGST1 activity with average IC(50) values of 9.1 and 265.4 muM, respectively. The nucleotide sequence reported in this paper was submitted to the GenBank Database with accession number DQ342327.

Cellular mechanisms of carbachol-stimulated Cl- secretion in rat epididymal epithelium.

Neurotransmitter-controlled Cl- secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca2+ activated Cl- channel blocker disulfonic acid stilbene (DIDS, 300 microM) only inhibited part of the CCH-induced Isc response, while nonselective Cl- channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 microM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca2+-chelating agent BAPTA/AM (50 microM) and an endoplasmic reticulum Ca2+ pump inhibitor, Thapsigagin (Tg, 1 microM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl- secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways.

Molecular cloning and characterization of cDNA encoding a novel cytosolic glutathione transferase from Clonorchis sinensis.

Glutathione transferases (GSTs) are a group of multifunction isoenzymes coded by many genes. A cDNA encoding a novel cytosolic GST enzyme was cloned from a Clonorchis sinensis (Cs) adult worm cDNA library by large-scale sequencing. This new cDNA contains 786 bp with a putative open reading frame of 212 amino acids. The deduced amino acid sequence exhibits 61% identity to C. sinensis cytosolic 28-kDa GST. Recombinant CsGST was overexpressed in Escherichia coli BL21(DE3) and was purified by Ni-NTA Agarose. Enzymatic assays showed that the recombinant CsGST had a high activity of 22.7 U mg(-1). The average K (m) of the CsGST for 1-chloro-2,4-dinitrobenzene is 111 microM. Cibacron blue F3GA and albendazole inhibited the CsGST activity with respective average IC(50) of 1.1 and 247.1 microM. It provides a model for the structure and physiological function analysis on CsGST. The nucleotide sequence reported in this paper has been submitted to the GenBank Database with accession number DQ179264.