Exogenous gibberellin delays maturation in persimmon fruit through transcriptional activators and repressors.

As the harvest season of most fruit is concentrated, fruit maturation manipulation is essential for the fresh fruit industry to prolong sales time. Gibberellin (GA), an important phytohormone necessary for plant growth and development, has also shown a substantial regulatory effect on fruit maturation; however, its regulatory mechanisms remain inconclusive. In this research, preharvest GA3 treatment effectively delayed fruit maturation in several persimmon (Diospyros kaki) cultivars. Among the proteins encoded by differentially expressed genes, 2 transcriptional activators (NAC TRANSCRIPTION FACTOR DkNAC24 and ETHYLENE RESPONSIVE FACTOR DkERF38) and a repressor (MYB-LIKE TRANSCRIPTION FACTOR DkMYB22) were direct regulators of GERANYLGERANYL DIPHOSPHATE SYNTHASE DkGGPS1, LYSINE HISTIDINE TRANSPORTER DkLHT1, and FRUCTOSE-BISPHOSPHATE ALDOLASE DkFBA1, respectively, resulting in the inhibition of carotenoid synthesis, outward transport of an ethylene precursor, and consumption of fructose and glucose. Thus, the present study not only provides a practical method to prolong the persimmon fruit maturation period in various cultivars but also provides insights into the regulatory mechanisms of GA on multiple aspects of fruit quality formation at the transcriptional regulation level.

Chalkophomycin Biosynthesis Revealing Unique Enzyme Architecture for a Hybrid Nonribosomal Peptide Synthetase and Polyketide Synthase.

Chalkophomycin is a novel chalkophore with antibiotic activities isolated from Streptomyces sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of N-hydroxylpyrrole, 2H-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the chm biosynthetic gene cluster from S. sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The chm cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 chm-type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.

Characterization of Chalkophomycin, a Copper(II) Metallophore with an Unprecedented Molecular Architecture.

Metals play essential roles in life by coordination with small molecules, proteins, and nucleic acids. Although the coordination of copper ions in many proteins and methanobactins is known, the coordination chemistry of Cu(II) in natural products and their biological functions remain underexplored. Herein, we report the discovery of a Cu(II)-binding natural product, chalkophomycin (CHM, 1), from Streptomyces sp. CB00271, featuring an asymmetric square-coordination system of a bidentate diazeniumdiolate and a conjugated 1H-pyrrole 1-oxide-oxazoline. The structure of 1 may inspire the synthesis of Cu(II) chelators against neurodegenerative diseases or Cu(II)-based antitumor therapeutics.

A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble.

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor ß (TGF-ß) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-ß family that form latent pro/mature domain complexes.

Determination of the Disulfide Structure of Murine Meteorin, a Neurotrophic Factor, by LC-MS and Electron Transfer Dissociation-High-Energy Collisional Dissociation Analysis of Proteolytic Fragments.

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC-MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials.

Investigating the Role of Artemin Glycosylation.

PURPOSE: Oligosaccharides play diverse and unpredictable functional roles when attached to proteins and are a largely unexplored scaffold for deconstructing and attributing novel functions to proteins during drug development. Here, the glycoprotein Artemin (ART) was carefully assessed by multiple analytical methods that allow us to provide a comprehensive understanding of how N-linked glycosylation impact the structural and functional properties of ART. METHODS: Modification of the N-linked glycan of ART was performed by incubation with various enzymes. Biological assays and systems were used to examine the relative activity and pharmacokinetic properties of ART as a function of glycosylation. In order to reveal the conformational impact of glycosylation on ART, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed in addition to differential scanning calorimetry. The colloidal stability of ART glycovariants was assessed by dynamic light scattering, viscometry, and solubility assays. RESULTS: No difference in pharmacokinetics or relative potency was revealed between glycosylated and nonglycosylated ART. Surprisingly, the HDX-MS data indicated that the glycan does not greatly influence the conformation and dynamics of the protein. In contrast, differences in thermal and colloidal stability clearly revealed a role of glycosylation in increasing the solubility and stability of ART. CONCLUSIONS: Our findings demonstrate how careful analysis using multiple advanced techniques can be used to identify and dissect the multiple potential functions of protein glycosylation and form a prerequisite for glycoengineering and drug development of glycoproteins.

Full-length transcriptome profiling for fruit development in Diospyros oleifera using nanopore sequencing.

OBJECTIVES: Diospyros oleifera, one of the most economically important Diospyros species, is an ideal model for studying the fruit development of persimmon. While, the lack of whole-transcriptome has hindered the complex transcriptional regulation mechanisms of sugar and tannin during fruit development. DATA DESCRIPTION: We applied Oxford Nanopore Technologies to six developmental stage of fruit from D. oleifera for use in transcriptome sequencing. As a result of full-length transcriptome sequencing, 55.87 Gb of clean data were generated. After mapping onto the reference genome of D. oleifera, 51,588 full-length collapsing transcripts, including 2,727 new gene loci and 43,223 transcripts, were obtained. Comprehensively annotated, 38,086 of new transcripts were functional annotation, and 972 lncRNAs, 7,159 AS events were predicted. Here, we released the transcriptome database of D. oleifera at different stage of fruit development,which will provide a fundamention of to investigatethe transcript structure, variants and evolution of persimmon.

Integrated Metabolomic-Transcriptomic Analysis Reveals Diverse Resource of Functional Ingredients From Persimmon Leaves of Different Varieties.

Persimmon leaves are used for making persimmon leaf tea or as functional ingredients due to their enrichment in flavonoids, the beneficial mineral contents, and favorable flavors contributed by volatile aroma compounds. The varieties/cultivars had a significant influence on the quality and flavor of persimmon leaf tea. In this study, the integrated metabolomic-transcriptomic analysis was conducted to investigate the potential in flavonoid biosynthesis, mineral absorption, and degradation of aromatic compounds from tender leaves of "Diospyros kaki. Heishi" (HS), "Diospyros kaki Thunb. Nishimurawase" (NM), and "Diospyros kaki Thunb. Taifu" (TF), using rootstock "Diospyros Lotus Linn" (DL) as the control. The metabolomic analysis showed that 382, 391, and 368 metabolites were differentially accumulated in the comparison of DL vs. HS, DL vs. NM, and DL vs. TF, respectively, and 229 common metabolites were obtained by comparative analysis. By RNA sequencing, 182,008 unigenes with 652 bp of mean length were annotated and 2,598, 3,503, and 3,333 differentially expressed genes (DEGs) were detected from the comparison of DL vs. HS, DL vs. NM, and DL vs. TF, respectively. After the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, 6, 6, and 3 DEGs [with | log2(fold change)| ≥ 1 simultaneously in the three comparisons] involved in flavonoid biosynthesis, mineral absorption, and degradation of aromatic compounds, respectively, were selected for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation and the consistent trends of the relative expression level of each DEG with RNA sequencing (RNA-seq) data were observed. Based on the transcriptomic analysis and qRT-PCR validation, it was observed that the leaves of HS, NM, and TF had the greatest level of mineral absorption, flavonoid biosynthesis, and degradation of aromatic compounds, respectively. In addition, a positive correlation between the 15 DEGs and their metabolites was observed by the conjoint analysis. Thus, the tender leaves of HS, NM, and TF could be recommended for the production of persimmon leaf tea rich in mineral elements, flavonoid, and aroma compounds, respectively.

Undescribed benzophenone and xanthones from cave-derived Streptomyces sp. CB09001.

A new benzophenone huanglongmycin (HLM) D (1) and two new monomeric xanthones huanglongmycin E (2) and F (3), together with four known aromatic polyketides aloesaponarin II (4) and the previously isolated huanglongmycin A-C (5-7) were obtained from cave-derived Streptomyces sp. CB09001. The structures of 1-3 were established based on 1D, 2D NMR and HRMS data. Compounds 1-7 may be biosynthesized by a type II huanglongmycin polyketide synthase based on gene inactivation of hlmG encoding KSɑ in hlm gene cluster and their plausible biosynthetic mechanism was proposed.

Characterization of the complete chloroplast genome sequence of Diospyros kaki cv. Luotiantianshi.

Diospyros kaki cv. Luotiantianshi is a rare germplasm of Diospyros Linn in the world. In this study, we generated the complete chloroplast (cp) genome of D. kaki cv. Luotiantianshi. The complete cp genome was 157,773 bp in length, containing a large single copy region (LSC) of 87,066 bp, a small single copy region (SSC) of 18,529 bp, and two inverted repeat (IR) regions of 26,089 bp. The new sequence has a total of 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Further, phylogenetic analysis showed that the D. kaki cv. Luotiantianshi has a close relationship with Diospyros kaki. This study provides important information for future evolution, genetic and molecular biology studies of Diospyros.

[The role of miRNA-122 expression during the acute liver failure in mice induced by D-GalN/LPS].

OBJECTIVE: To investigate the expression of miR-122 and its relationship with progression and development of acute liver failure in mice induced by D-GalN/LPS, and to explore new biomarker(s) for early diagnosis of acute liver failure. METHODS: BALB/C mice were randomly divided into four groups: the mice were given D-GalN (900 mg/kg body weight) and LPS (10 micog/kg body weight) intraperitoneally (i.p.) to construct the acute liver model; whereas the control groups were given D-GalN (900 mg/kg), LPS (10 microg/kg) and normal saline respectively. All biochemical and histological indexes were determined at 0, 1, 3, 5, 7 and 9 h respectively after administration. Real-time RT-PCR were used to detect the expression of miR-122 and pro-inflammatory cytokines, furthermore, the expression of miR-122 was verified by LNA (lock nucleic acid)-Northern-blot. ALT and AST levels were tested by biochemistry analyzer. Serum pro-inflammatory cytokine levels were tested by ELISA. RESULTS: The mortality rate was about 80% at 24h after D-GalN/LPS treatment, but no mortality was observed in the other three control groups. Liver special miRNA miR-122 was highly expressed in liver tissue of normal mice (ct is approximately equal to 14), it was up-regulated significantly (P = 0.013) at first hour after treatment then down-regulated according to the development of acute liver failure, the change was more obvious at 9 h (ct is approximately equal to 15, P = 0.002). ALT and AST levels increased obviously at 3h after treatment and reached peak at 7 hours then they were declined sharply. It was found that the expression of miR-122 was faster and more durable than ALT. Pro-inflammatory cytokines related to acute liver failure including TNFa and IL-6 were all up-regulated in serum as well as liver tissue (P less than 0.05). Correlation analysis showed that miR-122 had a negative correlation with ALT (correlation coefficients -0.505) and positive correlations with TNFa and IL-6 (correlation coefficients were 0.493 and 0.674 respectively). CONCLUSIONS: Liver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.

Functional activity of anti-LINGO-1 antibody opicinumab requires target engagement at a secondary binding site.

LINGO-1 is a membrane protein of the central nervous system (CNS) that suppresses myelination of axons. Preclinical studies have revealed that blockade of LINGO-1 function leads to CNS repair in demyelinating animal models. The anti-LINGO-1 antibody Li81 (opicinumab), which blocks LINGO-1 function and shows robust remyelinating activity in animal models, is currently being investigated in a Phase 2 clinical trial as a potential treatment for individuals with relapsing forms of multiple sclerosis (AFFINITY: clinical trial.gov number NCT03222973). Li81 has the unusual feature that it contains two LINGO-1 binding sites: a classical site utilizing its complementarity-determining regions and a cryptic secondary site involving Li81 light chain framework residues that recruits a second LINGO-1 molecule only after engagement of the primary binding site. Concurrent binding at both sites leads to formation of a 2:2 complex of LINGO-1 with the Li81 antigen-binding fragment, and higher order complexes with intact Li81 antibody. To elucidate the role of the secondary binding site, we designed a series of Li81 variant constructs that eliminate it while retaining the classic site contacts. These Li81 mutants retained the high affinity binding to LINGO-1, but lost the antibody-induced oligodendrocyte progenitor cell (OPC) differentiation activity and myelination activity in OPC- dorsal root ganglion neuron cocultures seen with Li81. The mutations also attenuate antibody-induced internalization of LINGO-1 on cultured cortical neurons, OPCs, and cells over-expressing LINGO-1. Together these studies reveal that engagement at both LINGO-1 binding sites of Li81 is critical for robust functional activity of the antibody.

Expression of angiotensin-converting enzyme 2 in CCL4-induced rat liver fibrosis.

The renin angiotensin system (RAS) plays a major role in liver fibrosis. A novel homologue of angiotensin converting enzyme, ACE2, was identified as a negative regulator of RAS as it degrades Ang II to Ang1-7. We investigated in vivo the expression of ACE2 in liver fibrosis. We evaluated the relationship between biochemical variables and liver tissue expression of ACE2, the correlation between a histological assessment of liver fibrosis and liver tissue expression of ACE2. Male SD rats were randomly divided into a CCL4 group which received injections of CCL4 and the control group which received injections of olive oil. Liver pathology was examined by H&E and Sirius red staining, and real-time PCR was performed to determine the gene expression levels of ACE2 and ACE. Real-time PCR analysis revealed that ACE2 mRNA was higher at the two-, four-, and six-week time points, respectively (p<0.01). Similarly, hepatic ACE mRNA was significantly increased after CCL4 injection. There was a significant correlation between ACE and ACE2 gene expression (r=0.750, P<0.001). ACE2 gene expression strongly correlated with ALT (r=0.669, P<0.0001) and AST levels (r=0.815, P<0.0001). There was a significant correlation between circulating ACE2 and histological scores of liver fibrosis. ACE2 and ACE gene expression correlated with the ISHAK score (r=0.850, P<0.001; r=0.806, P<0.001). There was a significant relationship between ACE2 gene expression and the degree of liver fibrosis. ACE2 plays a crucial role in liver fibrogenesis.

[Real-time quantification of microRNAs in Huh7 cells by stem-loop reverse transcriptase polymerase chain reaction].

OBJECTIVE: To establish a convenient realtime PCR which can detect microRNAs in the human hepatoma cell line, Huh7 cells. METHODS: Total RNAs in Huh7 cells were extracted. MicroRNA 122, 24 and 146a were assayed by microRNA array, and then verified by Northern blot. Stem-loop RT-PCR and poly(A)-tailed RT-PCR were used to detect the above microRNAs. Data were analyzed with Quantity One software and 7500 system software. RESULTS: Microarray signal intensity of microRNA 122, 24 and 146a in Huh7 cells was 2201.49, 410.20 and 4.70, whose relative expression was confirmed as 0.0383, 0.0249, 0.0001 through Northern blot. While the poly(A)-tailed RT-PCR might only measure microRNA 122, Stem-loop RT-PCR could detect microRNA 122, 24 and 146a, whose average dCt was 2.5, 5.8 and 12.1 in accordance with microRNA array and Northern blot. CONCLUSION: Stem-loop RT-PCR can specifically and sensitively quantity microRNA levels, regardless of their abundance.

The persimmon (Diospyros oleifera Cheng) genome provides new insights into the inheritance of astringency and ancestral evolution.

Persimmon (Diospyros kaki) is an oriental perennial woody fruit tree whose popular fruit is produced and consumed worldwide. The persimmon fruit is unique because of the hyperaccumulation of proanthocyanidins during fruit development, causing the mature fruit of most cultivars to have an astringent taste. In this study, we obtained a chromosome-scale genome assembly for 'Youshi' (Diospyros oleifera, 2n = 2x = 30), the diploid species of persimmon, by integrating Illumina sequencing, single-molecule real-time sequencing, and high-throughput chromosome conformation capture techniques. The assembled D. oleifera genome consisted of 849.53 Mb, 94.14% (799.71 Mb) of which was assigned to 15 pseudochromosomes, and is the first assembled genome for any member of the Ebenaceae. Comparative genomic analysis revealed that the D. oleifera genome underwent an ancient γ whole-genome duplication event. We studied the potential genetic basis for astringency development (proanthocyanidin biosynthesis) and removal (proanthocyanidin insolublization). Proanthocyanidin biosynthesis genes were mainly distributed on chromosome 1, and the clustering of these genes is responsible for the genetic stability of astringency heredity. Genome-based RNA-seq identified deastringency genes, and promoter analysis showed that most of their promoters contained large numbers of low oxygen-responsive motifs, which is consistent with the efficient industrial application of high CO2 treatment to remove astringency. Using the D. oleifera genome as the reference, SLAF-seq indicated that 'Youshi' is one of the ancestors of the cultivated persimmon (2n = 6x = 90). Our study provides significant insights into the genetic basis of persimmon evolution and the development and removal astringency, and it will facilitate the improvement of the breeding of persimmon fruit.

Effect of mesenchymal stem cells on Sjögren-like mice and the microRNA expression profiles of splenic CD4+ T cells.

Mesenchymal stem cells (MSCs) serve immuno-regulatory functions and offer a promising novel treatment for certain autoimmune diseases. The present study investigated the therapeutic effect of mice bone marrow (BM)-MSCs on mice with relatively late stage of Sjögren-like disease and the impact of BM-MSCs on the microRNA (miRNA) expression profiles of splenic CD4+ T cells. Female NOD/Ltj mice were randomized into two groups: The disease group (n=8) and the MSC-treated group (n=8). Female ICR mice served as the healthy control group (n=8). The MSC-treated group received an injection of MSCs when they were 26 weeks old. Water intake, blood glucose and salivary flow rate were measured and submandibular glands were resected and stained with hematoxylin and eosin to calculate the focus score. The concentrations of interleukin (IL)-2, IL-6, hepatocyte growth factor, interferon γ, IL-10, prostaglandin E2, transforming growth factor ß1 and tumor necrosis factor-α in serum were measured using ELISA. The expression of miRNAs in splenic CD4+ T cells were measured using deep sequencing. The results demonstrated that treatment with BM-MSCs prevented a decline in the salivary flow rate and lymphocyte infiltration in the salivary glands of NOD mice, indicating that MSC-treatment had a therapeutic effect on NOD mice with relatively late stage of Sjögren-like disease. ELISA and deep sequencing results showed that the three groups of mice had different serum concentrations of cytokines/growth factors and different miRNA expression profiles of splenic CD4+ T cells. This implies that the alteration in serum levels of cytokines/growth factors and miRNA expression profiles of splenic CD4+ T cells may explain the therapeutic effect MSCs have on Sjögren's syndrome.

Anti-hyperlipidemic properties of CM108 (a flavone derivative) in vitro and in vivo.

Peroxisome proliferator-activated receptors (PPARs) and liver X receptor alpha are ligand-activated transcription factors that belong to nuclear receptors superfamily and are involved in the regulation of lipid metabolism. PPAR, especially PPAR-alpha, PPAR-gamma agonists and liver X receptor alpha agonists can regulate the expression or biosynthesis of some factors involved in the formation and function of HDL, such as apolipoprotein (apo) A-I and ATP binding cassette transporter A1 (ABCA1). It is well known that HDL plays an important role in the treatment of hyperlipidemia as the carrier of reverse cholesterol transport. In the present study, the anti-hyperlipidemic properties of CM108, a derivative of flavone, 9-Hydroxy-2-mercapto-6-phenyl-2-thioxo-1,3,5-trioxa-2lambda(5)-phospha-cyclopenta[b]naphthalen-8-one, were studied. Through the transactivation assays of in vitro study, it was discovered that CM108 could activate PPAR-alpha PPAR-gamma and liver X receptor alpha at 40-150 microg/ml, which subsequently resulted in activating ABCA1 promoter and enhancing apoA-I and apoA-II production, whereas reducing apoC-III production significantly. Furthermore, after in vivo study that the hyperlipidemic rats were treated with CM108 for 4 weeks, a significant increase was found in HDL cholesterol levels (26.7%, P<0.05) and a significant decrease was also noticed in triglyceride levels (26.3%, P<0.01) at 100 mg/kg CM108 group compared with that of control animals. Meanwhile, the atherogenicity index, represented by total cholesterol/HDL ratio, was significantly reduced (P<0.01). In conclusion, CM108 can effectively elevate HDL levels and lower triglyceride levels in hyperlipidemic rats maybe by regulating a series of genes, receptors and proteins related to HDL.

Methylation profile of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma.

AIM: To establish the DNA methylation patterns of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma (HCC). METHODS: The methylation specific polymerase chain reaction in conjunction with sequencing verification was used to establish the methylation patterns of the 14 genes in the liver tissues of four healthy liver donors, as well as tumor and the paired non-cancerous tissues of 30 HCC patients. RESULTS: While 11 genes (ATP-binding cassette, sub-family G (WHITE), member 2(ABCG2), activating transcription factor (ATF2), beta-2-microglobulin (B2M), deoxycytidine kinase (DCK), occludin (OCLN), v-raf-1 murine leukemia viral oncogene homolog (RAF1), ralA binding protein 1 (RALBP1), splicing factor (45 kD) (SPF45), S-phase kinase-associated protein 2 (p45) (SKP2), tumor protein p53 (Li-Fraumeni syndrome) (TP53) and topoisomerase (DNA) II beta (TOP2B)) maintained the unmethylated patterns, three genes displayed to various extents the hypermethylation state in tumor tissues in comparison with the normal counterparts. The catalase (CAT) was hypermethylated in tumor and the neighboring non-cancerous tissue of one case (3.3%). Both glutathione S-transferase pi (GSTpi) (80%, 24/30 in tumor and 56.7%, 17/30 in the paired non-cancerous tissues) and cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) (CFTR) (77%, 23/30 in tumor and 50%, 15/30 in the paired non-cancerous tissues) genes were prevalently hypermethylated in HCC as well as their neighboring non-cancerous tissues. No significant difference in the hypermethylation occurrence was observed between the HCC and its neighboring non-cancerous tissues. CONCLUSION: Hypermethylation of promoter CpG islands of both CFTR and GSTpi genes occurs prevalently in HCC, which may correlate with the low expression of these two genes at the mRNA level and has the profound etiological and clinical implications. It is likely to be specific to the early phase of HCC carcinogenesis.

Inhibitory effects of antisense oligodeoxynucleotide on expression of vascular endothelia growth factor by human hepatocarcinoma cells.

BACKGROUND: It is widely recognized that the growth of solid tumor depends on angiogenesis. Vascular endothelia growth factor (VEGF) is an endothelial cell-specific mitogen that promotes angiogenesis in solid tumor. Inhibition of angiogenesis is considered a promising approach for cancer therapy, and treatments including administration of antisense drugs and RNA interference for the VEGF gene are geared to the suppression of tumor angiogenesis. METHODS: As a new approach for gene therapy of hepatocellular carcinoma (HCC), four groups of antisense oligodeoxynucleotide (ASODN) (A-Cap, A-AUG, A-UGA and A-Exon-3) were used to block the expression of VEGF, then VEGF mRNA and protein were detected by RT-PCR and Western blot. RESULTS: After treatment with ASODN, the relative VEGF mRNA levels of A-Cap, A-AUG, A-UGA, and A-Exon-3 were decreased significantly to (32+/-9)%, (63+/-1)%, (86+/-3)%, and (70+/-5)%, respectively(F=64.18, P<0.001). The relative VEGF protein levels of A-Cap, A-AUG, A-UGA and A-Exon-3 were decreased significantly to (41+/-5)%, (59+/-3)%, (88+/-7)%, and (79+/-9)% respectively (F=60.64, P<0.001). CONCLUSIONS: Among the four ASODNs, the ASODN for Cap structure showed the strongest inhibitory effect and that for A-UGA, the least (P<0.05 ). The inhibitory effect of ASODN on the expression of VEGF proteins was similar to that of VEGF mRNA expression.

[Inhibitory effects of antisense oligonucleotides on VEGF gene expression by human hepatocellular carcinoma cells].

OBJECTIVE: To investigate the inhibitory effects of antisense oligonucleotides to different sequences on VEGF gene expression by human hepatoma cells. METHODS: SMMC7721 cells were cultured under normoxic or hypoxic conditions for 24 h, followed by being transfected with different antisense oligonucleotides (A06513 to cap structure, A06514 to translation initiation, A06515 to Exon-3 and A06516 to translation terminal). The total RNAs from the cells were extracted and the VEGF expression were examined with RT-PCR. The relative concentrations of VEGF transcripts in SMMC772 cells from different groups were determined using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA as internal standard. RESULTS: In response to the hypoxic challenge, SMMC7721 cells upregulated VEGF mRNA; Comparative to the control (no oligonucleotides), A06513, A06514, A06515, and A06516 had obvious sequence-specific inhibitory effect on VEGF gene expression, with the ratio of VEGF over GAPDH of 0.49+/-0.08, 0.71+/-0.12, 0.72+/-0.11 and 0.86+/-0.12, respectively (F=12.21, P< 0.05). A06513 showed the strongest inhibitory effect (P<0.01). CONCLUSION: The antisense oligonucleotides complementary to VEGF cap structure, may become a potential alternative for antisense gene therapy of HCC.