Background: The human orphan receptor TLX (NR2E1) is a key regulator of neurogenesis, adult stem cell maintenance, and tumorigenesis. However, little is known about the genetic and transcriptomic events that occur following TLX overexpression in human cell lines.

Aims: Here, we used cytogenetics and RNA sequencing to investigate the effect of TLX overexpression with an inducible vector system in the HEK 293T cell line.

Methods and results: Conventional spectral karyotyping was used to identify chromosomal abnormalities, followed by fluorescence in situ hybridization (FISH) analysis on chromosome spreads to assess TLX DNA copy number. Illumina paired-end whole transcriptome sequencing was then performed to characterize recurrent genetic variants (single nucleotide polymorphisms (SNPs) and indels), expressed gene fusions, and gene expression profiles. Lastly, flow cytometry was used to analyze cell cycle distribution. Intriguingly, we show that upon transfection with a vector containing the human TLX gene (eGFP-hTLX), an isochromosome forms on the long arm of chromosome 6, thereby resulting in DNA gain of the TLX locus (6q21) and upregulation of TLX. Induction of the eGFP-hTLX vector further increased TLX expression levels, leading to G0-G1 cell cycle arrest, genetic aberrations, modulation of gene expression patterns, and crosstalk with other nuclear receptors (AR, ESR1, ESR2, NR1H4, and NR3C2). We identified a 49-gene signature associated with central nervous system (CNS) development and carcinogenesis, in addition to potentially cancer-driving gene fusions (LARP1-CNOT8 and NSL1-ZDBF2) and deleterious genetic variants (frameshift insertions in the CTSH, DBF4, POSTN, and WDR78 genes).

Conclusion: Taken together, these findings illustrate that TLX may play a pivotal role in tumorigenesis via genomic instability and perturbation of cancer-related processes.

Keywords: NR2E1; TLX; cancer; genome instability; nuclear receptors.

Nuclear Receptor subfamily 1, group H, member 4 (NR1H4) is a receptor for bile acids and has an important role in regulating energy metabolism in liver, muscle and adipose tissues in humans and animals. In this study, we cloned the full coding region of NR1H4 gene from porcine Longissimus dorsi by Rapid amplification of cDNA end (RACE). Results indicated that the open reading frame of NR1H4 covered 1461 bp encoding 486 amino acid residues and the deduced amino acid sequence was 91-94 % identical to that of Homo sapiens, Bos taurus, Macaca mulatta, Gorilla gorilla, and Ovis aries. Bioinformatic analysis indicated that NR1H4 contained 31 phosphorylation sites with 14 serine, 6 threonine and 11 tyrosine. One single nucleotide polymorphism (SNP) was detected by PCR-RFLP in 3' untranslated region of exon 9 (NR1H4) and the allele frequency analysis showed that A allele frequency was low among 396 pigs from five breeds. The NR1H4 mRNA expression pattern showed that NR1H4 gene was expressed highly in live and Longissimus dorsi. This work provided an important experimental basis for further research on mechanism of lipid metabolism and fat deposition in pigs.

Pigs are well recognized as a model for humans in research studies due to similarities in metabolism and physiology between the two species. The potential for pigs to model humans in studying metabolic diseases is highly dependent on similarities in hepatic metabolism between the two species, including similarities in the farnesoid X receptor (FXR; NR1H4) which regulate bile acid homeostasis. During initial cloning of porcine FXR (pFXR), an alternative splice variant (pFXR-SV1) was isolated which contained a four amino acid (MYTG) insert that exerted a dominant positive effect on the wild type receptor (pFXR-WT). The current study investigated the role of this insert in the dominant positive effect. Individual point mutations were made to the first three amino acids of the MYTG insert. Mutations of the methionine (M) or threonine (T) to alanine (A) reduced the dominant positive effect, while mutation of the tyrosine (Y) to either A or phenylalanine (F) completely abolished the dominant positive effect. Treatment with the tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4) increased the dominant positive effect of pFXR-SV1 by about 30%. These results suggest that the dominant positive effect may be dependent on the phosphorylation status of the tyrosine in the MYTG insert. The human variant hFXRα+ has the same MYTG insert as pFXR-SV1, but did not cause a dominant positive effect on hFXR-WT and significantly reduced the activity of hFXR-WT. Thus, although the MYTG insert is conserved in both human and pig, the effects of this insert are different in the two species.

Ethnopharmacological relevance: The well-known Chinese prescription, Xiaoyan Lidan Formula (XYLDF), possesses efficiency of heat-clearing, dampness-eliminating and jaundice-removing. It has long been used clinically for the treatment of hepatobiliary diseases due to intrahepatic cholestasis (IHC). However, the mechanism of XYLDF for its therapeutic effects remains elusive.

Aim of the study: The study aimed to explore the potential targets for liver protective mechanism of XYLDF based on network pharmacology and experimental assays in ANIT-induced cholestatic hepatic injury (CHI) in rats.

Materials and methods: On the basis of the 29 serum migrant compounds of XYLDF elucidated by UPLC-TOF-MS/MS, a network pharmacology approach was applied for the mechanism prediction. Systematic networks were constructed to identify potential molecular targets, biological processes, and signaling pathways. And the interactions between significantly potential targets and active compounds were simulated by molecular docking. For the mechanism validation, an ANIT-induced rat model was used to evaluate the effects of XYLDF on CHI according to serum biochemistry, bile flow rates, histopathological examination, and the gene and protein expression including enzymes related to synthesis, export, and import of bile acid in liver and ileum, and those of inflammatory cytokines, analyzed by RT-qPCR and WB.

Results: The results of network pharmacology research indicated TNF (TNF-α), RELA (NF-κB), NR1H4 (FXR), and ICAM1 (ICAM-1) to be the important potential targets of XYLDF for cholestatic liver injury, which are related to bile metabolism and NF-κB-mediated inflammatory signaling. And the molecular docking had pre-validated the prediction of network pharmacology, as the core active compounds of XYLDF had shown strong simulation binding affinity with FXR, followed by NF-κB, TNF-α, and ICAM-1. Meanwhile, the effects of XYLDF after oral administration on ANIT-induced CHI in rats exhibited the decreased levels of transaminases (ALT and AST), TBA, and TBIL in serum, raised bile flow rates, and markedly improved hepatic histopathology. Furthermore, consistent to the above targets prediction and molecular docking, XYLDF significantly up-regulated the expression of FXR, SHP, BSEP, and MRP2, and down-regulated CYP7A1 and NTCP in liver, and promoted expression of IBABP and OSTα/β in ileum, suggesting the activation of FXR-mediated pathway referring to bile acid synthesis, transportation, and reabsorption. Moreover, the lower levels of TNF-α in plasma and liver, as well as the reduced hepatic gene and protein expression of NF-κB, TNF-α, and ICAM-1 after XYLDF treatment revealed the suppression of NF-κB-mediated inflammatory signaling pathway, as evidenced by the inhibition of nuclear translocation of NF-κB.

Conclusions: XYLDF exhibited an ameliorative liver protective effect on ANIT-induced cholestatic hepatic injury. The present study has confirmed its mechanism as activating the FXR-regulated bile acid pathway and inhibiting inflammation via the NF-κB signaling pathway.

Keywords: Cholestatic Hepatic Injury; FXR; Inflammation; NF-κB; Network Pharmacology; XYLDF.

Many computational pipelines exist for the detection of differentially expressed genes. However, computational pipelines for functional gene detection rarely exist. We developed a new computational pipeline for functional gene identification from transcriptome profiling data. Key features of the pipeline include batch effect correction, clustering optimization by gap statistics, gene ontology analysis of clustered genes, and literature analysis for functional gene discovery. By leveraging this pipeline on RNA-seq datasets from two mouse retinal development studies, we identified 7 candidate genes involved in the formation of the photoreceptor outer segment. The expression of top three candidate genes (Pde8b, Laptm4b, and Nr1h4) in the outer segment of the developing mouse retina were experimentally validated by immunohistochemical analysis. This computational pipeline can accurately predict novel functional gene for a specific biological process, e.g., development of the outer segment and synapses of the photoreceptor cells in the mouse retina. This pipeline can also be useful to discover functional genes for other biological processes and in other organs and tissues.

Metabolic associated fatty liver disease (MAFLD) is associated with obesity, type 2 diabetes mellitus, and other metabolic syndromes. Farnesoid X receptor (FXR, NR1H4) plays a prominent role in hepatic lipid metabolism. This study combined the expression of liver genes in FXR knockout (KO) mice and MAFLD patients to identify new pathogenic pathways for MAFLD based on genome-wide transcriptional profiling. In addition, the roles of new target genes in the MAFLD pathogenic pathway were also explored. Two groups of differentially expressed genes were obtained from FXR-KO mice and MAFLD patients by transcriptional analysis of liver tissue samples. The similarities and differences between the two groups of differentially expressed genes were analyzed to identify novel pathogenic pathways and target genes. After the integration analysis of differentially expressed genes, we identified 134 overlapping genes, many of which have been reported to play an important role in lipid metabolism. Our unique analysis method of comparing differential gene expression between FXR-KO mice and patients with MAFLD is useful to identify target genes and pathways that may be strongly implicated in the pathogenesis of MAFLD. The overlapping genes with high specificity were screened using the Gene Expression Omnibus (GEO) database. Through comparison and analysis with the GEO database, we determined that BHMT2 and PKLR could be highly correlated with MAFLD. Clinical data analysis and RNA interference testing in vitro confirmed that BHMT2 may a new regulator of lipid metabolism in MAFLD pathogenesis. These results may provide new ideas for understanding the pathogenesis of MAFLD and thus provide new targets for the treatment of MAFLD.

Keywords: BHMT2; FXR; PPAR γ; metabolic associated fatty liver disease; transcription analysis.

Accumulating evidences suggested an association between gut microbiome dysbiosis and impaired glycemic control. Ginsenoside Rb1 (Rb1) is a biologically active substance of ginseng, which serves anti-diabetic effects. However, its working mechanism especially interaction with gut microbes remains elusive in detail. In this study, we investigated the impact of Rb1 oral supplementation on high fat diet (HFD) induced obesity mice, and explored its mechanism in regulating blood glucose. The results showed that higher liver weight and lower cecum weight were observed in HFD fed mice, which was maintained by Rb1 administration. In addition, Rb1 ameliorated HFD induced blood lipid abnormality and improved insulin sensitivity. Several mRNA expressions in the liver were measured by quantitative real-time PCR, of which UCP2, Nr1H4, and Fiaf were reversed by Rb1 treatment. 16S rRNA sequencing analysis indicated that Rb1 significantly altered gut microbiota composition and increased the abundance of mucin-degrading bacterium Akkermansia spp. compared to HFD mice. As suggested via functional prediction, amino acid metabolism was modulated by Rb1 supplementation. Subsequent serum amino acids investigation indicated that several diabetes associated amino acids, like branched-chain amino acids, tryptophan and alanine, were altered in company with Rb1 supplementation. Moreover, correlation analysis firstly implied that the circulation level of alanine was related to Akkermansia spp.. In summary, Rb1 supplementation improved HFD induced insulin resistance in mice, and was associated with profound changes in microbial composition and amino acid metabolism.

Keywords: amino acid metabolism; correlation analysis; ginsenoside Rb1; gut microbiota; insulin sensitivity.

Liver regeneration after extreme hepatocyte loss occurs via transdifferentiation of biliary epithelial cells (BECs), which includes dedifferentiation of BECs into bi-potential progenitor cells (BPPCs) and subsequent redifferentiation into nascent hepatocytes and BECs. Although multiple molecules and signaling pathways have been implicated to play roles in the BEC-mediated liver regeneration, mechanisms underlying the dedifferentiation-redifferentiation-transition and the early phase of BPPC redifferentiation that is pivotal for both hepatocyte and BEC directions, remain largely unknown. The zebrafish extreme liver damage model, genetic mutation, pharmacological inhibition, transgenic lines, whole-mount and fluorescent in situ hybridizations and antibody staining, single-cell RNA sequencing, quantitative real-time PCR, and heat shock-inducible overexpression were used to investigate roles and mechanisms of farnesoid X receptor (FXR, encoded by nr1h4) in regulating BPPC redifferentiation. The nr1h4 expression was significantly upregulated in response to extreme liver injury. Genetic mutation or pharmacological inhibition of FXR was ineffective to BEC-to-BPPC dedifferentiation, but blocked the redifferentiation of BPPCs to both hepatocytes and BECs, leading to accumulation of undifferentiated or less-differentiated BPPCs. Mechanistically, induced overexpression of extracellular signal-related kinase 1 (ERK1, encoded by mapk3) rescued the defective BPPC-to-hepatocyte redifferentiation in the nr1h4 mutant, and ERK1 itself was necessary for the BPPC-to-hepatocyte redifferentiation. The Notch activities in the regenerating liver of nr1h4 mutant attenuated, and induced Notch activation rescued the defective BPPC-to-BEC redifferentiation in the nr1h4 mutant. CONCLUSIONS: FXR regulates BPPC-to-hepatocyte and BPPC-to-BEC redifferentiations through ERK1 and Notch, respectively. Given recent applications of FXR agonists in the clinical trials for liver diseases, this study proposes potential underpinning mechanisms by characterizing roles of FXR in the stimulation of dedifferentiation-redifferentiation-transition and BPPC redifferentiation.

Keywords: FXR; biliary epithelial cell; extreme liver damage; hepatocyte; transdifferentiation.

Background: We have recently reported a mouse model of PNAC in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of canalicular bile acid transporter Abcb11, bilirubin exporter Abcc2, and sterol transporters Abcg5/8. The aim of this study was to examine the role of TNFα in promoting PNAC in mice.

Methods: (1) Recombinant TNFα was administered to mice as well as in hepatocyte cell culture. (2) Tnfr1/2^-/- or WT mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN). (3) WT/DSS-PN mice were also infused with infliximab at 10mg/kg on days 3 and 10 of PN. PNAC was defined by increased serum AST, ALT, total bile acids and bilirubin.

Results: Intraperitoneal injection of TNFα into WT mice or TNFα treatment of Huh7 hepatocarcinoma cells and primary mouse hepatocytes suppressed mRNA transcription of bile (Abcb11[BSEP], Abcc2[MRP2]) and sterol transporters (Abcg5, Abcg8) and their regulators Nr1h3(FXR) and Nr1h4(LXR). DSS-PN mice with PNAC had increased hepatic TNFα mRNA expression and significant reduction of mRNA expression of Abcb11, Abcc2, Abcg5/8, Nr1h3 and Nr1h4. In contrast, PNAC development was prevented, and mRNA expression normalized, in both Tnfr1/2^-/- /DSS-PN mice and DSS-PN mice treated with infliximab.

Conclusions: TNFα is a key mediator in the pathogenesis of PNAC through suppression of hepatocyte Abcb11, Abcc2 and Abcg5/8. Pharmacologic targeting of TNFα as a therapeutic strategy for PNAC thus deserves further investigation. This article is protected by copyright. All rights reserved.

Keywords: Cholestasis; Infliximab; Intestinal failure associated liver disease; Tumor Necrosis Factor alpha; hepatic macrophages.

Background: Adrenarche reflects the developmental growth of the adrenal zona reticularis, which produces increasing adrenal androgen secretion (e.g., DHEA/DHEAS) from ages ~5-15 years. We hypothesized that the study of the genetic determinants associated with variations in serum DHEAS during adrenarche might detect genetic variants influencing the rate or timing of this process.

Subjects and methods: Genome-wide genotyping was performed in participants of the Chilean pediatric GOCS cohort (n=788). We evaluated the genetic determinants of DHEAS levels at genome-wide level and in targeted genes associated with steroidogenesis. To corroborate our findings, we evaluated a polygenic risk score for age at pubarche, based upon the discovered variants, in children from the same cohort.

Results: We identified one significant variant at the genome-wide level in the full cohort, close to the GALR1 gene (P = 3.81x10 -8). In addition, variants suggestive of association (P <1x10 -5) were observed in PRLR, PITX1, PTPRD, NR1H4, and BCL11B. Stratifying by sex, we found variants suggestive of association in SERBP1 and CAMTA1/VAMP3 for boys and near ZNF98, TRPC6, and SULT2A1 for girls. We also found significant reductions in age at pubarche in those children with higher polygenic risk scores for greater DHEAS based on these newly identified variants.

Conclusions: Our results disclose one variant associated with DHEAS concentrations at the level of GWAS significance, and several variants with suggestive association, which may be involved in the genetic regulation of adrenarche.

Keywords: DHEAS; GWAS; adrenarche; genomics; human genetics; polygenic risk score; pubarche.

Background and aims: Parenteral nutrition (PN) associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1β derived from activated hepatic macrophages to suppress hepatocyte FXR signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model.

Methods: To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sodium sulfate for 4 days) followed by central venous catheterization and 14 day-infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, biliary and sterol transporter expression were determined.

Results: GW4064 (30mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis, reversed the suppressed mRNA expression of Nr1h4/FXR, Abcb11/BSEP, Abcc2, Abcb4, and Abcg5/8, and normalized serum bile acids. Chromatin-immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll1b, Ccr2, Itgam, Ly6C), and hepatic macrophage cytokine transcription in response to LPS in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1β exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15 and Ostα) were not different among groups, supporting a liver specific effect of GW4064 in this model CONCLUSION: GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling resulting in increased expression of canalicular bile, sterol and phospholipid transporters, and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.

Keywords: GW4064; IL-1β; Intestinal failure associated liver disease; Liver X receptor; bile acids.

We report a newborn who presented with multiple limb and facial anomalies, endocrine disorders, and progressively worsening low-GGT cholestasis. A liver biopsy revealed hepatocellular cholestasis with giant cell transformation. Immunohistochemical staining revealed complete absence of BSEP protein compared to control liver. A large 2q24-32.2 deletion leading to loss of 78 OMIM genes. Multiple structural anomalies, epilepsy and endocrine anomalies have been described with hemizygous loss of these genes. This deletion also resulted in complete heterozygous deletion of ABCB11, which encodes the bile salt export pump (BSEP). Genetic analysis did not reveal any pathogenic variants, deletions, or duplications in the other ABCB11 allele. A heterozygous variant in NR1H4, which causes the autosomal recessive progressive familial intrahepatic cholestasis type 5, was also detected. The possible explanations for the PFIC type 2 phenotype in heterozygous loss of ABCB11 include genetic modifiers or di-genic disease with a compound ABCB11 deletion and an NR1H4 missense variant; or undetected pathogenic variants in the other ABCB11 or NR1H4 alleles.

Keywords: congenital heart disease; congenital hypothyroidism; dysmorphology; farnesoid X-activated receptor; metabolic liver; neonatal cholestasis.

Background: Diet-induced obesity (DIO) and psychological stress are significant independent regulators of gastrointestinal physiology; however, our understanding of how these two disorders influence the host-microbe interface is still poorly characterized. The aim of this study was to assess the combined influences of diet-induced obesity and psychological stress on microbiome composition and colonic gene expression.

Methods: C57BL/6J mice (n = 48) were subject to a combination of 22 weeks of Western diet (WD) feeding and a chronic restraint stressor (CRS) for the last 4 weeks of feeding. At the end of the combined intervention, microbiome composition was determined from cecal contents, and colonic tissue gene expression was assessed by multiplex analysis using NanoString nCounter System and real-time qPCR.

Results: WD feeding induced a DIO phenotype with increased body weight, worsened metabolic markers, and alterations to microbiome composition. CRS reduced body weight in both dietary groups while having differential effects on glucose metabolism. CRS improved the Firmicutes/Bacteroidetes ratio in WD-fed animals while expanding the Proteobacteria phyla. Significantly lower expression of colonic Tlr4 (p = 0.008), Ocln (p = 0.004), and Cldn3 (p = 0.004) were noted in WD-fed animals compared to controls with no synergistic effects observed when combined with CRS. No changes to colonic expression of downstream inflammatory mediators were observed. Interestingly, higher levels of expression of Cldn2 (p = 0.04) and bile acid receptor Nr1h4 (p = 0.02) were seen in mice exposed to CRS.

Conclusion: Differential but not synergistic effects of WD and CRS were noted at the host-microbe interface suggesting multifactorial responses that require further investigation.

Keywords: chronic stress; colonic gene expression; diet-induced obesity; gut microbiome; intestinal permeability; tight junction proteins; western diet.

NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.

Keywords: CRISPR/Cas9; HepaRG; NADPH cytochrome P450 reductase; bile acid metabolism; cholesterol biosynthesis; proteomics.

Background: Early-stage lung squamous cell carcinoma (LUSC) progression is accompanied by changes in immune microenvironments and the expression of immune-related genes (IRGs). Identifying innate IRGs associated with prognosis may improve treatment and reveal new immunotherapeutic targets.

Methods: Gene expression profiles and clinical data of early-stage LUSC patients were obtained from the Gene Expression Omnibus and The Cancer Genome Atlas databases and IRGs from the InnateDB database. Univariate and multivariate Cox regression and LASSO regression analyses were performed to identify an innate IRG signature model prognostic in patients with early-stage LUSC. The predictive ability of this model was assessed by time-dependent receiver operator characteristic curve analysis, with the independence of the model-determined risk score assessed by univariate and multivariate Cox regression analyses. Overall survival (OS) in early-stage LUSC patients was assessed using a nomogram and decision curve analysis (DCA). Functional and biological pathways were determined by gene set enrichment analysis, and differences in biological functions and immune microenvironments between the high- and low-risk groups were assessed by ESTIMATE and the CIBERSORT algorithm.

Results: A signature involving six IRGs (SREBF2, GP2, BMX, NR1H4, DDX41, and GOPC) was prognostic of OS. Samples were divided into high- and low-risk groups based on median risk scores. OS was significantly shorter in the high-risk than in the low-risk group in the training (P < 0.001), GEO validation (P = 0.00021) and TCGA validation (P = 0.034) cohorts. Multivariate Cox regression analysis showed that risk score was an independent risk factor for OS, with the combination of risk score and T stage being optimally predictive of clinical benefit. GSEA, ESTIMATE, and the CIBERSORT algorithm showed that immune cell infiltration was higher and immune-related pathways were more strongly expressed in the low-risk group.

Conclusion: A signature that includes these six innate IRGs may predict prognosis in patients with early-stage LUSC.

Keywords: early-stage lung squamous cell carcinoma; gene signature; immune cell infiltration; innate immune-related genes; prognosis; risk score.

Farnesoid X receptor (FXR, NR1H4) is a ligand-activated nuclear receptor, which regulates bile acid, lipid and glucose metabolism. Due to these functions, FXR has been investigated as a potential drug target for the treatment of liver diseases, such as primary biliary cholangitis and non-alcoholic steatohepatitis. Based on the previously described four splice variants, it has been suggested that alternative promoter usage and splicing may have an impact on total FXR activity as a result of encoding functionally diverse variants. Here we aimed for a systematic analysis of human hepatic FXR splice variants. In addition to the previously described FXRα1-4, we identified four novel splice variants (FXRα5-8) in human hepatocytes, which resulted from previously undetected exon skipping events. These newly identified isoforms displayed diminished DNA binding and impaired transactivation activities. Isoform FXRα5, which suppressed the transactivation activity of the functional isoform FXRα2, was further characterized as deficient in heterodimerization, coactivator recruitment and ligand binding. These findings were further supported by molecular dynamics simulations, which offered an explanation for the behavior of this isoform on the molecular level. FXRα5 exhibited low uniform expression levels in nearly all human tissues. Our systematic analysis of FXR splice variants in human hepatocytes resulted in the identification of four novel FXR isoforms, which all proved to be functionally deficient, but one novel variant, FXRα5, also displayed dominant negative activity. The possible associations with and roles of these novel isoforms in human liver diseases require further investigation.

Keywords: Alternative splicing; DNA binding; Dominant negative protein; Farnesoid X receptor; Nuclear receptor; Transactivation.

The aim of this study was to consider the expression of farnesoid X receptor (Fxr), liver X receptor (LXRα) and sirtuin 1 (Sirt1), oxidative stress, inflammation, apoptosis, and the protective role of N-acetylcysteine (NAC) in the liver of rats treated with cadmium (Cd). 30 Wistar rats were divided into 5 groups: G1 (control), G2 (single dose of Cd), G3 (continuous dose of Cd), G4 (single dose of Cd + continuous dose of NAC), and G5 (continuous dose of Cd + continuous dose of NAC). The apoptosis of hepatic cells was measured using the TUNEL assay. Levels of malondialdehyde (MDA), IL-10, TNF-α, and total antioxidant capacity (TAC) were measured by specific kits. The expression of Fxr, LXRα, and Sirt1 genes and ratio of Bax/Bcl2 was considered using RT-PCR. While NAC treatment improved TAC and IL-10 values, it decreased MDA and TNF-α levels in the liver of rats exposed to Cd (P < 0.001). NAC decreased Bax/Bcl2 in the liver of G4 and G5 groups (P < 0.001). Exposure to a continuous dose of Cd decreased Fxr, LXRα, and Sirt1 expression by 36.65- (P < 0.001), 12.52- (P < 0.001) and 11.34-fold (P < 0.001) compared to control, respectively. NAC increased Fxr, LXRα, and Sirt1 expression (P < 0.01) and decreased Cd concentrations in both serum and tissue samples in G4 and G5 groups. Our results suggested that NAC protects liver tissue against Cd toxicity by elevating antioxidant capacity, mitigating oxidative stress, inflammation, apoptosis and up-regulation of FXR, LXR, and SIRT1 genes.

Keywords: Antioxidants; Apoptotic genes; Cadmium; N-Acetyl cysteine; Oxidative stress.

Background: The dysregulation of microRNAs (miRNAs) and hepatotoxicity due to the aberrant accumulation of bile acids (BAs) are notorious causes that predispose an individual to the development of hepatocellular carcinoma (HCC). Farnesoid X receptor (FXR), encoded by NR1H4 gene, has been identified as a crucial BA receptor to maintain the homeostasis of BA pool and its expression is decreased in HCC. miR-382-5p plays an important role in the pathogenesis of many human malignancies and was reported to promote the proliferation and differentiation of normal liver cells and liver regeneration. However, there is still some controversy about its role in HCC microenvironment. This study aims to explore the expression pattern of miR-382-5p in HCC and its role in regulating FXR during the development of HCC.

Methods: Tissues collected from 30 HCC patients were subjected to extraction of total RNA and quantitative real-time PCR (qRT-PCR) for the analyses of miR-382-5p expression and NR1H4 mRNA levels, and their expressions were verified by analyzing the online HCC-related GSE datasets. The role of miR-382-5p in regulating cellular proliferation and expression of FXR in different HCC cell lines was analyzed by qRT-PCR, Western Blot, real-time cellular analysis (RTCA) and luciferase reporter assays. The role of miR-382-5p in regulating downstream genes of FXR in HCC cells was also analyzed.

Results: miR-382-5p was upregulated in HCC tissues and inversely associated with the downregulation of NR1H4 mRNA levels. The luciferase reporter assay proved that miR-382-5p directly targeted the 3'-untranslated region (3'-UTR) of human NR1H4 mRNA. Overexpression of miR-382-5p led to a malignant proliferation of HCC cells by suppressing the expression of FXR. In contrast, blocking the endogenous miR-382-5p was sufficient to suppress the cellular proliferation rate of HCC through increasing FXR expression. Additionally, miR-382-5p inhibited the expression of some target genes of FXR, including SHP, FGF19 and SLC51A, and this inhibitory effect was FXR-dependent.

Conclusion: Therefore, miR-382-5p promotes the progression of HCC in vitro by suppressing FXR and could serve as a valuable therapeutic target for HCC treatment.

Keywords: FXR; hepatocellular carcinoma; miRNA-382-5p; proliferation.

Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of disorders characterized by defects in bile secretion and presentation with intrahepatic cholestasis in infancy or childhood. The most common types include PFIC 1 (deficiency of FIC1 protein, ATP8B1 gene mutation), PFIC 2 (bile salt export pump deficiency, ABCB11 gene mutation), and PFIC 3 (multidrug resistance protein-3 deficiency, ABCB4 gene mutation). Mutational analysis of subjects with normal gamma-glutamyl transferase cholestasis of unknown etiology has led to the identification of newer variants of PFIC, known as PFIC 4, 5, and MYO5B related (sometimes known as PFIC 6). PFIC 4 is caused by the loss of function of tight junction protein 2 (TJP2) and PFIC 5 is due to NR1H4 mutation causing Farnesoid X receptor deficiency. MYO5B gene mutation causes microvillous inclusion disease (MVID) and is also associated with isolated cholestasis. Children with TJP2 related cholestasis (PFIC-4) have a variable spectrum of presentation. Some have a self-limiting disease, while others have progressive liver disease with an increased risk of hepatocellular carcinoma. Hence, frequent surveillance for hepatocellular carcinoma is recommended from infancy. PFIC-5 patients usually have rapidly progressive liver disease with early onset coagulopathy, high alpha-fetoprotein and ultimately require a liver transplant. Subjects with MYO5 B-related disease can present with isolated cholestasis or cholestasis with intractable diarrhea (MVID). These children are at risk of worsening cholestasis post intestinal transplant (IT) for MVID, hence combined intestinal and liver transplant or IT with biliary diversion is preferred. Immunohistochemistry can differentiate most of the variants of PFIC but confirmation requires genetic analysis.

Keywords: Biliary diversion; Hepatocellular carcinoma; Microvillous inclusion disease; Progressive familial intrahepatic cholestasis; Tight junction protein.

Background: Type 2 diabetes mellitus (T2DM) is a heterogeneous, metabolic, and chronic condition affecting vast numbers of the world's population. The related variables and T2DM associations have not been fully understood due to their diverse nature. However, functional genomics can facilitate understanding of the disease. This information will be useful in drug design, advanced diagnostic, and prognostic markers.

Aim: To understand the genetic causes of T2DM, this study was designed to identify the differentially expressed genes (DEGs) of the disease.

Methods: We investigated 20 publicly available disease-specific cDNA datasets from Gene Expression Omnibus (GEO) containing several attributes including gene symbols and clone identifiers, GenBank accession numbers, and phenotypic feature coordinates. We analyzed an integrated system-level framework involving Gene Ontology (GO), protein motifs and co-expression analysis, pathway enrichment, and transcriptional factors to reveal the biological information of genes. A co-expression network was studied to highlight the genes that showed a coordinated expression pattern across a group of samples. The DEGs were validated by quantitative PCR (qPCR) to analyze the expression levels of case and control samples (50 each) using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene.

<strong class="sub-title"> Results: </strong> From the list of 50 DEGs, we ranked three T2DM-related genes (<i>p</i> < 0.05): <i>SRR</i>, <i>NFKB1</i>, and <i>PDE4B</i>. The enriched terms revealed a significant functional role in amino acid metabolism, signal transduction, transmembrane and intracellular transport, and other vital biological functions. <i>DMBX1</i>, <i>TAL1</i>, <i>ZFP161</i>, <i>NFIC</i> (66.7%), and <i><em>NR1H4</em></i> (33.3%) are transcriptional factors associated with the regulatory mechanism. We found substantial enrichment of insulin signaling and other T2DM-related pathways, such as valine, leucine and isoleucine biosynthesis, serine and threonine metabolism, adipocytokine signaling pathway, P13K/Akt pathway, and Hedgehog signaling pathway. The expression profiles of these DEGs verified by qPCR showed a substantial level of twofold change (FC) expression <math><mrow><mo>(</mo> <msup><mn>2</mn> <mrow> <msub><mrow><mo>-</mo> <mi>Δ</mi> <mi>Δ</mi> <mtext>C</mtext></mrow> <mtext>T</mtext></msub> </mrow> </msup> <mo>)</mo></mrow> </math> in the genes <i>SRR</i> (FC ≤ 0.12), <i>NFKB1</i> (FC ≤ 1.09), and <i>PDE4B</i> (FC ≤ 0.9) compared to controls (FC ≥ 1.6). The downregulated expression of these genes is associated with pathophysiological development and metabolic disorders.

Conclusion: This study would help to modulate the therapeutic strategies for T2DM and could speed up drug discovery outcomes.

Keywords: T2DM; cDNA datasets; differential expressed genes; enrichment analysis; expression profiling; qPCR.

Background & aims: Progressive familial intrahepatic cholestasis (PFIC) is a collective term for a heterogenous group of rare, inherited cholestasis syndromes. The number of genes underlying the clinical PFIC phenotype is still increasing. While progressive liver disease and its sequelae such as portal hypertension, pruritus, and hepatocellular carcinoma determine transplant-free survival, extrahepatic manifestations may cause relevant morbidity.

Methods: We performed a literature search for extrahepatic manifestations of PFIC associated with pathogenic gene variants in ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, and MYO5B. To illustrate the extrahepatic symptoms described in the literature, PFIC cases from our centers were revisited.

Results: Extrahepatic symptoms are common in PFIC subtypes, where the affected gene is expressed at high levels in other tissues. While most liver-associated complications resolve after successful orthotopic liver transplantation (OLT), some extrahepatic symptoms show no response or even worsen after OLT.

Conclusion: The spectrum of extrahepatic manifestations in PFIC highlights essential, non-redundant roles of the affected genes in other organs. Extrahepatic features contribute towards low health-related quality of life (HRQOL) and morbidity in PFIC. While OLT is often the only remaining, curative treatment, potential extrahepatic manifestations need to be carefully monitored and addressed.