[Differential analysis of gene expression profiles in hepatocellular carcinoma patients with high and low levels of alpha-fetoprotein].

Objective: To investigate the differential gene expression profiles of alpha-fetoprotein (AFP) high- and low-expressing hepatocellular carcinoma (HCC), and to provide a theoretical basis for the molecular mechanism and prognosis analysis of HCC. Methods: The transcriptome data and related clinical information from 368 HCC cases were obtained from the Cancer Gene Atlas (TCGA) public database. The samples were divided into AFP high expression (AFP(high)) group and low expression (AFP(low)) group according to the quartile of AFP mRNA expression, with 92 cases in each group. The differential gene analysis was carried out using the DEseq2 package in the R software. The functional and KEGG pathway enrichment analysis of the differential genes was performed using ClusterProfiler package. The protein-protein interaction network was constructed to screen hub genes using the String database and Cytoscape software. The single-sample GSEA analysis was performed to enrich and score signature gene sets using the GSVA package. And then RNAseq data and real-time quantitative polymerase chain reaction (RT-qPCR) were used for independent dataset validation and tissue validation. Results: The clinical analysis showed that high expression of AFP was significantly associated with poor pathological differentiation and ethnicity (P<0.05 for both). A total of 1 382 differential genes were obtained by bioinformatics analysis, of which 931 genes were up-regulated and 451 genes were down-regulated in AFP(high) group. GO enrichment analysis showed that the highly expressed genes were mainly correlated with the processes of appendage development, limb development, and skeletal system development, while lowly expressed genes were related to metabolic-related processes such as xenobiotic metabolism, steroid metabolism, and cellular response to xenobiotic stimuli. KEGG pathway enrichment analysis revealed that highly expressed genes were mainly involved in primary immunodeficiency, neuroactive ligand-receptor interaction, and cytokine-cytokine receptor interaction, while lowly expressed genes were mainly involved in retinol metabolism, chemical carcinogenesis, steroid hormone biosynthesis and other pathways. A prognostic related gene set that was consisted of AURKB, TTK, CENPA, UBE2C, HJURP, and KIF15 was identified. And the high expression of this gene set was related to the shorter recurrence-free survival and overall survival time in HCC patients, and its enrichment score was positively correlated with AFP expression (r=0.475, P<0.001). The validation results of RNAseq data were basically consistent with the TCGA data. The RT-qPCR results showed that AURKB, KIF15, and UBE2C were significantly overexpressed in HCC tissues with high AFP expression. Although the expression of AURKB, TTK, KIF15, and UBE2C was not related to recurrence-free survival and overall survival of HCC patients, there was a tendency that the patients with high AFP levels showed relatively shorter recurrence-free survival time and overall survival time. Conclusions: There is a large difference in gene expression profiles between AFP(high) and AFP(low) HCC. The prognostic signature may cooperate with AFP to promote the initiation and development of HCC. It also may explain the tumorigenesis in HCC with different AFP levels, and provide new clues for the prognosis of HCC.

NAG-1/GDF15 accumulates in the nucleus and modulates transcriptional regulation of the Smad pathway.

Protein dynamics, modifications and trafficking are all processes that can modulate protein activity. Accumulating evidence strongly suggests that many proteins have distinctive roles dependent on cellular location. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a transforming growth factor-ß (TGF-ß) superfamily protein that has a role in cancer, obesity and inflammation. NAG-1 is synthesized and cleaved into a mature peptide, which is ultimately secreted into the extracellular matrix (ECM). In this study, we have found that full-length NAG-1 is expressed in not only the cytoplasm and ECM, but also in the nucleus. NAG-1 is dynamically moved to the nucleus, exported into cytoplasm and further transported into the ECM. We have also found that nuclear NAG-1 contributes to inhibition of the Smad pathway by interrupting the Smad complex. Overall, our study indicates that NAG-1 is localized in the nucleus and provides new evidence that NAG-1 controls transcriptional regulation in the Smad pathway.

Catalpol: a potential therapeutic for neurodegenerative diseases.

Neurodegenerative disorders, e.g., Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by the progressive loss of neurons and subsequent cognitive decline. They are mainly found in older populations. Due to increasing life expectancies, the toll inflicted upon society by these disorders continues to become heavier and more prominent. Despite extensive research, however, the exact etiology of these disorders is still unknown, though the pathophysiological mechanisms have been attributed to oxidative, inflammatory and apoptotic injury in the brain. Moreover, there is currently no promising therapeutic agent against these neurodegenerative changes. Catalpol, an iridoid glucoside contained richly in the roots of the small flowering plant species Rehmannia glutinosa Libosch, has been shown to have antioxidation, anti-inflammation, anti-apoptosis and other neuroprotective properties and plays a role in neuroprotection against hypoxic/ischemic injury, AD and PD in both in vivo and in vitro models. It may therefore represent a potential therapeutical agent for the treatment of hypoxic/ischemic injury and neurodegenerative diseases. Based on our studies and those of others in the literature, here we comprehensively review the role of Catalpol in neuroprotection against pathological conditions, especially in neurodegenerative states and the potential mechanisms involved.

Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We aimed to discover biomarkers in urinary exosomes to detect acute kidney injury (AKI), which has a high mortality and morbidity. Animals were injected with cisplatin. Urinary exosomes were isolated by differential centrifugation. Protein changes were evaluated by two-dimensional difference in gel electrophoresis and changed proteins were identified by mass spectrometry. The identified candidate biomarkers were validated by Western blotting in individual urine samples from rats subjected to cisplatin injection; bilateral ischemia and reperfusion (I/R); volume depletion; and intensive care unit (ICU) patients with and without AKI. We identified 18 proteins that were increased and nine proteins that were decreased 8 h after cisplatin injection. Most of the candidates could not be validated by Western blotting. However, exosomal Fetuin-A increased 52.5-fold at day 2 (1 day before serum creatinine increase and tubule damage) and remained elevated 51.5-fold at day 5 (peak renal injury) after cisplatin injection. By immunoelectron microscopy and elution studies, Fetuin-A was located inside urinary exosomes. Urinary Fetuin-A was increased 31.6-fold in the early phase (2-8 h) of I/R, but not in prerenal azotemia. Urinary exosomal Fetuin-A also increased in three ICU patients with AKI compared to the patients without AKI. We conclude that (1) proteomic analysis of urinary exosomes can provide biomarker candidates for the diagnosis of AKI and (2) urinary Fetuin-A might be a predictive biomarker of structural renal injury.

Thromboxanes: synthase and receptors.

Thromboxane A2 is a biologically potent arachidonate metabolite through the cyclooxygenase pathway. It induces platelet aggregation and smooth muscle contraction and may promote mitogenesis and apoptosis of other cells. Its roles in physiological and pathological conditions have been widely documented. The enzyme that catalyzes its synthesis, thromboxane A2 synthase, and the receptors that mediate its actions, thromboxane A2 receptors, are the two key components critical for the functioning of this potent autacoid. Recent molecular biological studies have revealed the structure-function relationship and gene organizations of these proteins as well as genetic and epigenetic factors modulating their gene expression. Future investigation should shed light on detailed molecular signaling events specifying thromboxane A2 actions, and the genetic underpinning of the enzyme and the receptors in health and disease.

Genomic organization, chromosomal localization, and expression of the murine thromboxane synthase gene.

Thromboxane synthase (TS) is a membrane-bound cytochrome P450 enzyme catalyzing the synthesis of TxA2, a potent modulator of vascular smooth muscle contraction and platelet aggregation. TS plays an important role in hemostasis and may be intimately involved in the etiology of cardiovascular, renal, and immune diseases. Restriction enzyme mapping, subcloning, and DNA sequencing analysis of recombinant phage lambda and P1 clones revealed that exons encoding the 1.9-kb mouse TS mRNA are dispersed over >150 kb genomic DNA. Determination of the intron-exon splicing junctions established that the mouse TS gene (Tbxas1) is encoded by 13 exons ranging in size from 53 (exon III) to 315 bp (exon IX). Genomic Southern analysis and fluorescence in situ hybridization suggested that the gene is a single-copy gene, located on chromosome 6 near the midpoint between the centromere and the Igkappa gene. An alternatively spliced variant of the Tbxas1 transcript, lacking the exon XII-encoded sequence, has been detected in normal mouse tissues. Ribonuclease protection and 5'-RACE assays identified at least five major transcription start sites clustered within 31 bp of the Tbxas1 promoter. The 5'-most start site is not preceded by a TATA box, suggesting transcription can be initiated in a TATA-independent manner. Transfection analyses indicated that the expression of Tbxas1 is controlled by a short (70-bp) positive regulatory sequence and several upstream repressive elements. Mutational studies further demonstrated that NF-E2/AP-1 and Sp1 exerted activating and repressive, respectively, effects on the promoter. These studies provide the genetic tools and information for TS research in mice, which should expedite understanding of the genetic contribution of TS in normal physiology as well as in disease states.

Multiple factors regulating the expression of human thromboxane synthase gene.

Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, approximately 75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (approximately 55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.

Genomic structure and polymorphism of the human thromboxane synthase-encoding gene.

Thromboxane synthase (TS) is a cytochrome P-450 (CYP450) enzyme catalyzing the conversion of prostaglandin endoperoxide (PGH2) into thromboxane A2 (TxA2) which plays a crucial role in hemostasis and cardiovascular diseases. Twelve genomic clones containing the DNA encoding the human TS gene (hTS) were isolated and characterized to determine the exon/intron boundaries and restriction maps of the nearly contiguous structure of the gene. The hTS contains 13 exons spanning more than 150 kb. Its first five exons, divided by relatively large introns, spread over 100 kb, but encode less than one third of the full-length TS transcript. Southern analysis indicates that the human haploid genome contains a single copy of the TS gene. Although multiple transcription start points (tsp) are utilized, transcription of hTS is primarily TATA-independent, as determined by promoter-directed reporter gene expression in transfected cells. A dinucleotide (CA) repetitive sequence identified in the ninth intron of the gene exhibits allelic polymorphism. At least four distinctive alleles, containing from 13 to 20 copies of the CA repeats, have been detected.

Cloning and characterization of the human thromboxane synthase gene promoter.

A genomic phage clone hybridized to the 5' end of human thromboxane synthase (TS) cDNA was isolated. Sequencing analysis of a 1.7 kb subfragment revealed that it contained the entire 5' untranslated region and 46 bp of the coding sequence of TS cDNA, an upstream canonical TATA box (TATAAA), and several binding sites for transcription factors (AP1, PEA3, PU.1, and GR), indicative of a promoter/first exon region of the TS gene. RNase protection assay mapped the transcription start site of the human TS gene to the nucleotide A 30 bp downstream from the TATA box. The authenticity of the promoter was further confirmed by its ability to direct expression of a CAT reporter gene in transfected HL60 cells.

The porcine thromboxane synthase-encoding cDNA: sequence, mRNA expression and enzyme production in Sf9 insect cells.

A full-length cDNA encoding porcine thromboxane synthase (TS) was isolated and sequenced. The open reading frame encodes a 534-amino acid (aa) protein (M(r) 60,451) which shares more than 75% identity with TS from other species and is 30% homologous to several enzymes of the cytochrome P-450 III family. Sequence comparison among porcine (p), human (h), and murine (m) TS indicated conservation of eight Cys residues and one putative N-glycosylation site. Several highly conserved regions were identified at the near N terminus, middle and C terminus. The most divergent region lies at aa residues 290-325, within which a Lys308 residue was unique to pTS. Between aa residues 70 and 90, considerable divergence was observed in mTS. Northern analysis showed that the pTS gene was expressed as a 2.3-kb transcript primarily in lung, kidney and thymus. A high-titer recombinant (re-) baculovirus containing pTS cDNA was developed to conduct a time course study of enzyme production in Spodoptera frugiperda (Sf9) cells. TS activity was detectable in the microsomes of Sf9 cells 12-h post-infection and reached maximum by 48 h. The produced TS resembles purified pTS in catalysis, as well as inhibition by a substrate analog inhibitor.

Expression of two different forms of cDNA for thromboxane synthase in insect cells and site-directed mutagenesis of a critical cysteine residue.

cDNA coding for human placental thromboxane synthase (EC 5.3.99.5) was amplified by PCR from a human placental cDNA library and sequenced. This cDNA and a shorter cDNA isolated from a human lung cDNA library with a deletion of 163 bp near the 3' end were expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The cDNA from human placenta was expressed as an active enzyme (60 kDa) with a specific activity higher than those reported from other cell types, whereas the shorter cDNA was expressed in an inactive form (52 kDa). The active recombinant enzyme appeared to be unglycosylated as the molecular mass and the enzyme activity were not altered in the presence of tunicamycin. Site-directed mutagenesis was performed to convert a cysteine at position 480 in thromboxane synthase to a serine. This cysteine is found to be highly conserved in related cytochrome P-450 enzymes. The mutant enzyme was found to be inactive, although Western blot, immunoprecipitation and SDS/PAGE analysis indicated that the mutant enzyme was expressed at a level comparable with the wild-type enzyme. These results suggest that Cys-480 is essential for the enzyme catalytic activity and that the short-form cDNA may be a non-functional transcript.

Molecular cloning and expression of murine thromboxane synthase.

The complementary DNA (cDNA) for murine thromboxane synthase (TS) was isolated from a lung cDNA library. The full-length cDNA (1,910 bp) encodes a 533 amino acid protein (Mr 58,220) sharing 78% identity with human TS. Sequence comparison indicated that one of the two N-glycosylation sites, eight of the eleven cysteine residues, and a heme-binding domain are conserved in both murine and human TS sequences. The authenticity of the cDNA was confirmed by transient expression of a catalytically active TS in cos-1 cells. Northern analysis indicated that murine TS gene was expressed primarily in lung, kidney, and spleen. Interestingly, the size of mRNA in the kidney was approximately 100 to 150 bp shorter than that in the lung or spleen (2.2 Kb). RT-PCR and restriction mapping indicated that neither the coding sequence nor the 3' untranslated region could account for the observed size difference, suggesting a shorter 5' untranslated region and/or poly (A) tail in the kidney transcript.

Mapping of the human thromboxane synthase gene (TBXAS1) to chromosome 7q34-q35 by two-color fluorescence in situ hybridization.

Thromboxane synthase (TS) catalyzes the conversion of the prostaglandin endoperoxide into thromboxane A2 (TxA2), a potent vasoconstrictor and inducer of platelet aggregation. In concert with prostacyclin TxA2 plays a pivotal role in the maintenance of hemostasis. Deficiency of platelet TS activity has been shown to result in bleeding disorders. The potent effect of TxA2 on platelet function and vascular activity suggests a possible involvement of TS in normal and pathophysiological conditions such as cardiovascular disease. To aid in establishing the correlation of TS to disease states, we localized the human TS gene (TBXAS1) to chromosome 7q34-q35 using dual-color fluorescence in situ hybridization.

Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice.

The hepatitis B virus X protein acts as a transcriptional transactivator in vitro. To elucidate possible biological effects of X protein on liver cells in vivo, we generated four lines of transgenic mice carrying the X gene open reading frame under the control of the human alpha-1-antitrypsin regulatory region. The plasmid construct used to introduce the transgene was shown to encode a 16-kDa X protein with transactivating capability. The expression of X protein was detectable in liver tissue of transgenic animals of three of the lines by immunoblot analysis; levels of expression were highest in the first month after birth of the animals. Over 80 animals from the expressing lines were examined histologically. Most transgenic mice, some of which were observed for up to 2 years, remained normal. However, a few transgenic animals developed mild focal hepatitis, nuclear pleomorphism, focal necrosis, and/or nodular hyperplasia in the liver. Increased mitotic activity of hepatocytes also was observed. We conclude that, at the level of expression achieved in these transgenic mice, the hepatitis B virus transcriptional transactivator X protein alone does not appear to mediate the development of serious liver damage or hepatocellular carcinomas.

Development of a transgenic mouse system for the analysis of stages in liver carcinogenesis using tissue-specific expression of SV40 large T-antigen controlled by regulatory elements of the human alpha-1-antitrypsin gene.

alpha-1-Antitrypsin (AAT) is the major antiprotease in human plasma; it is synthesized primarily in hepatocytes and to a lesser extent in several nonhepatic tissues. Under the control of regulatory elements of the human AAT gene, expression of SV40-large tumor antigen (T-ag) in transgenic mice occurred in the liver, stomach, pancreas, and kidney. Among seven founder transgenic animals, six developed liver carcinoma, four showed gastric neoplasia, and one developed pancreatic carcinoma. In three animals the kidneys showed glomerular or tubular epithelial hyperplasia but no malignancy. A stable transgenic line, 1812, was established. Members of this line reproducibly develop liver tumors by 10 weeks of age but do not exhibit any phenotypic changes in other tissues. Histological changes leading to liver tumor formation occurred with predictable kinetics and could be classified into four distinct stages: (a) embryonal/fetal stage, no recognizable histological changes; (b) newborn to 2 weeks of age, hyperplastic hepatocytes with reduced amounts of cytoplasm but no nuclear alterations; (c) between 3 and 8 weeks of age, diffuse liver cell dysplasia without observable tumor nodules; and (d) 8 weeks of age and thereafter, hepatocellular carcinomas in a background of liver dysplasia. Embryonic and newborn liver tissue showed uniform, high level expression of T-ag in the majority of hepatocytes by immunohistochemistry, whereas the dysplastic and tumoral stages were characterized by considerable variation in both the intensity of T-ag staining and the proportion of T-ag-positive cells. Immunoprecipitation analyses showed that T-ag was complexed with cellular protein p53 in all tumor samples. This study showed that SV40 T-ag expression in the liver resulted in cellular hyperplasia and dysplasia; additional event(s) apparently were required for progression to neoplasia. Those cooperating events occurred with predictable kinetics. This transgenic mouse system displays several similarities with human liver disease and provides a practical model for the study of separate steps in hepatocarcinogenesis.

Genetic control of human alpha-1-antitrypsin.

Alpha-1-antitrypsin (AAT) is the predominant protease inhibitor in human sera. The major physiological role of this inhibitor is to protect elastin fibers in the alveolar structure of the lung from excessive degradation by neutrophil elastase. AAT is synthesized predominantly by hepatocytes, although the AAT gene is expressed to a small degree in the epithelial cells of various tissues. Recent studies have shown that the enhanced liver-specific expression of the AAT gene is controlled by the binding of hepatic nuclear proteins to specific DNA sequences upstream from the structural gene. A variety of mutations within the AAT gene have been identified that result in a partial deficiency or total absence of the inhibitor in sera. Inheritance of a particular combination of these alleles can result in a predisposition towards the development of destructive lung disease. Interestingly, the most common AAT deficiency variant, designated PiZ, causes the mutant protein to accumulate as an insoluble aggregate within the lumen of the hepatic rough endoplasmic reticulum, which is an etiological agent for the development of liver disease. Overall, investigation into the genetic control of AAT has led to an increased understanding of the factors that control hepatic gene expression, as well as mechanisms involved in the pathophysiology of emphysema and liver cirrhosis.

Tissue-specific regulation of human alpha 1-antitrypsin gene expression in transgenic mice.

The 5'-flanking sequence of the human alpha 1-antitrypsin (AAT) gene contains multiple cis-regulatory elements, including a distal enhancer and proximal sequences essential for its transcription in cultured hepatoma cells. To understand better the promoter specificity of the AAT gene in vivo, transgenic mice harboring the AAT-SV40 hybrid promoter or the natural AAT promoter fused to a reporter gene (CAT) were generated. Examination of CAT activity in various tissues indicated that the CAT gene was expressed primarily in the liver and also, to a lesser extent, in tissues known to express the AAT gene. In addition, the cis-acting elements of the human AAT gene were utilized to drive the transcription of the SV40 T antigen gene in transgenic mice. Hepatocellular malignancy was found in all founder animals examined, while sporadic occurrence of malignancy was also observed in stomach, pancreas, and kidney. These results verify that the 5'-flanking region of the human AAT gene contains cis-regulatory elements sufficient to confer tissue specificity in vivo.

Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes.

Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. We report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human alpha 1-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating form the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the alpha 1-antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene.

The human alpha-1-antitrypsin (AAT) gene is expressed in the liver, and its deficiency causes pulmonary emphysema. We have demonstrated that its 5'-flanking region contains cis-acting elements capable of directing proper transcription in the presence of rat liver nuclear extract. The in vitro transcription system is tissue-specific in that the AAT promoter is functional in nuclear extracts prepared from the liver but not from HeLa cells. Experiments in which rat liver and HeLa nuclear extracts were mixed suggested the presence of a specific activator(s) in hepatocytes rather than a repressor(s) in nonproducing cells. Two protected regions were detected in the promoter by DNase I footprinting analysis with rat liver nuclear extracts. Region one spanned -78 to -52 and region two spanned -125 to -100 in the 5'-flanking sequence of the gene. By gel retardation assays with synthetic oligonucleotides, at least two distinct liver nuclear factors were identified, HNF-1 and HNF-2 (hepatocyte nuclear factors), which bound specifically to the first and second region, respectively. We present evidence that HNF-1 and HNF-2 are positively acting, tissue-specific transcription factors that regulate hepatic expression of the human AAT gene.