The Enigmas of Tissue Closure: Inspiration from Drosophila.

Hollow structures are essential for development and physiological activity. The construction and maintenance of hollow structures never cease throughout the lives of multicellular animals. Epithelial tissue closure is the main strategy used by living organisms to build hollow structures. The high diversity of hollow structures and the simplicity of their development in Drosophila make it an excellent model for the study of hollow structure morphogenesis. In this review, we summarize the tissue closure processes in Drosophila that give rise to or maintain hollow structures and highlight the molecular mechanisms and distinct cell biology involved in these processes.

Correlation between the RNA Expression and the DNA Methylation of Estrogen Receptor Genes in Normal and Malignant Human Tissues.

Estrogen plays a multifaceted function in humans via interacting with the estrogen receptors ERα, ERß, and G protein-coupled estrogen receptor 1 (GPER1). Previous research has predominantly concentrated on elucidating the signaling route of estrogen. However, the comprehensive understanding of the expression profile and control of these estrogen receptors in various human tissues is not well known. In the present study, the RNA levels of estrogen receptors in various normal and malignant human tissues were retrieved from the human protein atlas, the cancer genome atlas (TCGA), and the genotype-tissue expression (GTEx) databases for analyzing the expression profile of estrogen receptors through gene expression profiling interactive analysis (GEPIA). The status of DNA methylation of estrogen receptor genes from TCGA were analyzed through the software Wanderer and cBioPortal. The MethSurv tool was utilized to estimate the relevance between specific cytosine-guanine (CG) methylation and tumor survival. The expression profile analysis revealed that ERα, ERß, and GPER1 have unique expression patterns in diverse tissues and malignancies. The interesting results were the higher expression of ERß RNA in the male testis than in females and the positive association between the RNA level of ERα and the androgen receptor in different human normal tissues. Especially, the significant changes in GPER1 expression in multiple malignancies showed a consistent decrease with no exception, which indicates the role of GPER1 in common tumor inhibition. The finding on the expression profile provides clues for exploring novel potential physiological and pathophysiological functions of estrogen. The DNA methylation analysis manifested that the expression of GPER1 and ERα showed a substantial correlation with the methylation of specific CG sites in the cis-regulating region of the gene. However, no such association was observed for ERß. When comparing tumor tissues to normal tissues, the DNA methylation of certain CG sites of estrogen receptors showed a correlation with tumor survival but did not always correlate with the expression of that gene or with the expression of DNA methyltransferases. We proposed that the variation in DNA methylation at different CG sites in estrogen receptor genes had other functions beyond its regulatory role in its gene expression, and this might be associated with the progression and therapy efficiency of the tumor based on the modulation of the chromatin configuration.

Therapeutic potential of natural antisense transcripts and various mechanisms involved for clinical applications and disease prevention.

Antisense transcription, a prevalent occurrence in mammalian genomes, gives rise to natural antisense transcripts (NATs) as RNA molecules. These NATs serve as agents of diverse transcriptional and post-transcriptional regulatory mechanisms, playing crucial roles in various biological processes vital for cell function and immune response. However, when their normal functions are disrupted, they can contribute to human diseases. This comprehensive review aims to establish the molecular foundation linking NATs to the development of disorders like cancer, neurodegenerative conditions, and cardiovascular ailments. Additionally, we evaluate the potential of oligonucleotide-based therapies targeting NATs, presenting both their advantages and limitations, while also highlighting the latest advancements in this promising realm of clinical investigation.Abbreviations: NATs- Natural antisense transcripts, PRC1- Polycomb Repressive Complex 1, PRC2- Polycomb Repressive Complex 2, ADARs- Adenosine deaminases acting on RNA, BDNF-AS- Brain-derived neurotrophic factor antisense transcript, ASOs- Antisense oligonucleotides, SINEUPs- Inverted SINEB2 sequence-mediated upregulating molecules, PTBP1- Polypyrimidine tract binding protein-1, HNRNPK- heterogeneous nuclear ribonucleoprotein K, MAPT-AS1- microtubule-associated protein tau antisense 1, KCNQ1OT- (KCNQ1 opposite strand/antisense transcript 1, ERK- extracellular signal-regulated kinase 1, USP14- ubiquitin-specific protease 14, EGF- Epidermal growth factor, LSD1- Lysine Specific Demethylase 1, ANRIL- Antisense Noncoding RNA in the INK4 Locus, BWS- Beckwith-Wiedemann syndrome, VEGFA- Vascular Endothelial Growth component A.

CDC20 Holds Novel Regulation Mechanism in RPA1 during Different Stages of DNA Damage to Induce Radio-Chemoresistance.

Targeting CDC20 can enhance the radiosensitivity of tumor cells, but the function and mechanism of CDC20 on DNA damage repair response remains vague. To examine that issue, tumor cell lines, including KYSE200, KYSE450, and HCT116, were utilized to detect the expression, function, and underlying mechanism of CDC20 in radio-chemoresistance. Western blot and immunofluorescence staining were employed to confirm CDC20 expression and location, and radiation could upregulate the expression of CDC20 in the cell nucleus. The homologous recombination (HR) and non-homologous end joining (NHEJ) reporter gene systems were utilized to explore the impact of CDC20 on DNA damage repair, indicating that CDC20 could promote HR repair and radio/chemo-resistance. In the early stages of DNA damage, CDC20 stabilizes the RPA1 protein through protein-protein interactions, activating the ATR-mediated signaling cascade, thereby aiding in genomic repair. In the later stages, CDC20 assists in the subsequent steps of damage repair by the ubiquitin-mediated degradation of RPA1. CCK-8 and colony formation assay were used to detect the function of CDC20 in cell vitality and proliferation, and targeting CDC20 can exacerbate the increase in DNA damage levels caused by cisplatin or etoposide. A tumor xenograft model was conducted in BALB/c-nu/nu mice to confirm the function of CDC20 in vivo, confirming the in vitro results. In conclusion, this study provides further validation of the potential clinical significance of CDC20 as a strategy to overcome radio-chemoresistance via uncovering a novel role of CDC20 in regulating RPA1 during DNA damage repair.

Different Expression Pattern of G Protein-Coupled Estrogen Receptor GPER1 in Esophageal Squamous Cell Carcinoma and Adenocarcinoma.

Esophageal carcinoma is a male-dominant malignancy worldwide, and esophageal adenocarcinoma (EAC) shows more significant sex bias than esophageal squamous cell carcinoma (ESCC) in morbidity and mortality. The G protein-coupled estrogen receptor 1 (GPER1) is involved in several sex-related cancers; however, its expression level in esophageal carcinoma has been poorly investigated and its role is not precisely defined, depending on histological types. In the present study, the mRNA levels of GPER1 in esophageal carcinoma were collected from GEPIA and Oncomine databases for meta-analyses. The protein expression levels of GPER1 were detected by immunohistochemistry in the tissue microarray of EAC and ESCC. The GPER1 selective agonist G1, antagonist G15, and siRNA were applied in vitro to investigate their impacts on esophageal cell lines. Analysis of the RNA levels from the databases showed a decreased expression of GPER1 in overall esophageal carcinoma, and low expression levels of GPER1 were found to be associated with low survival of tumor patients. However, in the subgroup of EAC and its precancerous lesion, Barrett's esophagus, overexpression of GPER1 RNA was increased when compared with the normal tissues. The average staining scores of GPER1 protein in the tissue microarray of EAC were significantly higher than normal esophageal samples, and the rate of positive staining increased with the grade of poor tumor differentiation. The scores of GPER1 protein in ESCC tissues were lower than those in the normal tissues. The results from cell line experiments in vitro showed that the GPER1 agonist G1 inhibited proliferation and promoted apoptosis of ESCC cells EC109 with positive expression of GPER1. G1 had no obvious effect on normal esophageal NE2 cells with weak expression of GPER1. In addition, GPER1 RNA knockdown and application of antagonist G15 reversed the effects of G1 on EC109. The results of this study indicate that the expression levels of GPER1 are higher in EAC than in ESCC, which might be correlated with the dimorphic estrogen signaling pathway in different types of esophageal carcinoma.

Estradiol regulates the expression of CD45 splicing isoforms in lymphocytes.

CD45, a common leukocyte antigen expressed on the surface of all nucleated hematopoietic cells, indicates the developmental stage and functional status of lymphocytes by its alternative splicing isoforms. Estrogen is correlated with the immune activity of lymphocytes and is involved in the sex bias of several human autoimmune diseases, but the effect of estrogen on the expression of the CD45 splicing isoforms remains unknown. In the present study, a potential estrogen response element was identified on the opposite strand of the CD45 gene by bioinformatics software prediction. The results from RT-qPCR results showed that the expression levels of CD45RO isoform and CD45 antisense RNA were increased after the lymphocytes were treated with 10 nM 17beta-estradiol, and this effect of 17beta-estradiol was reversed when the lymphocytes were cotreated with an estrogen receptor antagonist. Moreover, bisulfite sequencing PCR showed that CD45 DNA methylation in lymphocytes was increased after the treatment with 10 nM 17beta-estradiol. In conclusion, estradiol regulated the expression of CD45 in an estrogen receptor-dependent manner and was associated with CD45 antisense RNA and DNA methylation. The results helped elucidate the regulatory mechanism of the expression of CD45 isoforms and the correlation between estrogen levels and immune activity in females.

AS1DHRS4, a head-to-head natural antisense transcript, silences the DHRS4 gene cluster in cis and trans.

The human genome, like other mammalian genomes, encodes numerous natural antisense transcripts (NATs) that have been classified into head-to-head, tail-to-tail, or fully overlapped categories in reference to their sense transcripts. Evidence for NAT-mediated epigenetic silencing of sense transcription remains scanty. The DHRS4 gene encodes a metabolic enzyme and forms a gene cluster with its two immediately downstream homologous genes, DHRS4L2 and DHRS4L1, generated by gene duplication. We identified a head-to-head NAT of DHRS4, designated AS1DHRS4, which markedly regulates the expression of these three genes in the DHRS4 gene cluster. By pairing with ongoing sense transcripts, AS1DHRS4 not only mediates deacetylation of histone H3 and demethylation of H3K4 in cis for the DHRS4 gene, but also interacts physically in trans with the epigenetic modifiers H3K9- and H3K27-specific histone methyltransferases G9a and EZH2, targeting the promoters of the downstream DHRS4L2 and DHRS4L1 genes to induce local repressive H3K9me2 and H3K27me3 histone modifications. Furthermore, AS1DHRS4 induces DNA methylation in the promoter regions of DHRS4L2 by recruiting DNA methyltransferases. This study demonstrates that AS1DHRS4, as a long noncoding RNA, simultaneously controls the chromatin state of each gene within the DHRS4 gene cluster in a discriminative manner. This finding provides an example of transcriptional control over the multiple and highly homologous genes in a tight gene cluster, and may help explain the role of antisense RNAs in the regulation of duplicated genes as the result of genomic evolution.

Beyond Genes: Epiregulomes as Molecular Commanders in Innate Immunity.

The natural fastest way to deal with pathogens or danger signals is the innate immune system. This system prevents too much inflammation and tissue damage and efficiently eliminates pathogens. The epiregulome is the chromatin structure influenced by epigenetic factors and linked to cis-regulatory elements (CREs). The epiregulome helps to end the inflammatory response and also assists innate immune cells to show specific action by making cell-specific gene expression patterns. This inspection unfolds two concepts: (1) how epiregulomes are shaped by switching the expression levels of genes, manoeuvre enzyme activity and earmark of chromatin modifiers on specific genes; during and after the infection, and (2) how the expression of specific genes (aids in prompt management of innate cell growth, or the reaction to aggravation and illness) command by epiregulomes that formed during the above process. In this review, the consequences of intrinsic immuno-metabolic remodelling on epiregulomes and potential difficulties in identifying the master epiregulome that regulates innate immunity and inflammation have been discussed.

ResNet incorporating the fusion data of RGB & hyperspectral images improves classification accuracy of vegetable soybean freshness.

The freshness of vegetable soybean (VS) is an important indicator for quality evaluation. Currently, deep learning-based image recognition technology provides a fast, efficient, and low-cost method for analyzing the freshness of food. The RGB (red, green, and blue) image recognition technology is widely used in the study of food appearance evaluation. In addition, the hyperspectral image has outstanding performance in predicting the nutrient content of samples. However, there are few reports on the research of classification models based on the fusion data of these two sources of images. We collected RGB and hyperspectral images at four different storage times of VS. The ENVI software was adopted to extract the hyperspectral information, and the RGB images were reconstructed based on the downsampling technology. Then, the one-dimensional hyperspectral data was transformed into a two-dimensional space, which allows it to be overlaid and concatenated with the RGB image data in the channel direction, thereby generating fused data. Compared with four commonly used machine learning models, the deep learning model ResNet18 has higher classification accuracy and computational efficiency. Based on the above results, a novel classification model named ResNet-R &H, which is based on the residual networks (ResNet) structure and incorporates the fusion data of RGB and hyperspectral images, was proposed. The ResNet-R &H can achieve a testing accuracy of 97.6%, which demonstrates a significant enhancement of 4.0% and 7.2% compared to the distinct utilization of hyperspectral data and RGB data, respectively. Overall, this research is significant in providing a unique, efficient, and more accurate classification approach in evaluating the freshness of vegetable soybean. The method proposed in this study can provide a theoretical reference for classifying the freshness of fruits and vegetables to improve classification accuracy and reduce human error and variability.

Human NRDRB1, an alternatively spliced isoform of NADP(H)-dependent retinol dehydrogenase/reductase enhanced enzymatic activity of benzil.

AIMS: Human NRDRB1, a 226 amino acid alternatively spliced isoform of the NADP(H)- dependent retinol dehydrogenase/reductase (NRDR), lacks the complete coding region of exon 3, but preserves all the important functional motifs for NRDR catalytic activity. Nevertheless, its tissue distribution and physiological function remain to be elucidated. METHODS: Expression of NRDRB1 and NRDR in cells and tissues was analyzed by semi-quantitative polymerase chain reaction (PCR) and western blot. NRDRB1 was expressed as a His(6) fusion protein and subjected to kinetics assays. RESULTS: Recombinant NRDRB1 had 1.2 to 8.6 fold higher k(cat)/K(m) values than recombinant NRDR, depending on the substrate. NRDRB1 catalyzed the NADPH-dependent reduction of α-dicarbonyl compounds, such as isatin, 9,10-phenanthrenequinone, and especially benzil. The significantly high catalytic activity and the relatively high expression in human liver of NRDRB1 conferred cellular resistance to benzil-induced cell toxicity and over-expression of NRDRB1 in low expressing Ec109 cells significantly enhanced cell tolerance toward benzil. CONCLUSIONS: Based on its substrate specificity, catalytic activity and relatively high expression in human liver tissue, our results suggest that NRDRB1, an alternatively spliced isoform of NRDR in vivo functions better than NRDR as a dicarbonyl reductase for xenobiotics containing reactive carbonyls. Our study is the first reporting this phenomenon of the enzymes involved in biochemical reactions.

The aberrant expression of CD45 isoforms and levels of sex hormones in systemic lupus erythematosus.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a common autoimmune disease with significant gender bias in women, and sex hormones are considered to play an important role in the regulation of immune activity. The CD45 isoforms generated through alternative splicing of mRNA identify different functional status of lymphocytes and also are suggested as a biomarker for assessing the progression of SLE, while the modulation of CD45 expression in SLE patients is not clear. METHODS: In this study, the peripheral blood sera of 46 SLE patients and 15 health individuals were collected for detecting the levels of sex hormones and immune associated factors. The expression of CD45 isoforms and the status of CD45 DNA methylation of the peripheral mononuclear blood cells were detected by flow cytometry and bisulfite sequencing PCR, respectively. RESULTS: The levels of complement C3 and IgA decreased, especially decline of the serum IgA to the level of selective immunoglobulin A deficiency, and the C-reactive protein increased in SLE patients when compared with healthy controls, which manifested the abnormal immune activity of the SLE patients. Sex hormones detection showed a decreased testosterone and increased prolactin in SLE. An accelerated expression of CD45RO, reduced CD45RA and CD45RB, and a relative hypermethylation of CD45 DNA in SLE were also identified that provided a clue to explain the possible regulatory mechanism for the immune function in SLE. CONCLUSION: The results indicated that the aberrant CD45 isoforms, DNA methylation and hormone levels might be correlated with the imbalanced immune activity of SLE patients. Key Points • Selective immunoglobulin A deficiency was significantly higher in SLE than in healthy individuals. • SLE patients had decreased testosterone and increased prolactin in the sera. • An aberrant expression of CD45 isoforms and CD45 DNA methylation were identified in SLE.

Deep Learning and Hyperspectral Images Based Tomato Soluble Solids Content and Firmness Estimation.

Cherry tomato (Solanum lycopersicum) is popular with consumers over the world due to its special flavor. Soluble solids content (SSC) and firmness are two key metrics for evaluating the product qualities. In this work, we develop non-destructive testing techniques for SSC and fruit firmness based on hyperspectral images and the corresponding deep learning regression model. Hyperspectral reflectance images of over 200 tomato fruits are derived with the spectrum ranging from 400 to 1,000 nm. The acquired hyperspectral images are corrected and the spectral information are extracted. A novel one-dimensional (1D) convolutional ResNet (Con1dResNet) based regression model is proposed and compared with the state of art techniques. Experimental results show that, with a relatively large number of samples our technique is 26.4% better than state of art technique for SSC and 33.7% for firmness. The results of this study indicate the application potential of hyperspectral imaging technique in the SSC and firmness detection, which provides a new option for non-destructive testing of cherry tomato fruit quality in the future.

Characterization of apoptosis and proliferation in esophageal carcinoma EC109 cells following siRNA-induced down-regulation of TRAF6.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an activator of the NF-κB transcription factor. NF-κB is involved in a variety of inflammatory, anti-apoptotic, and gene regulatory pathways and was recently found to be over-expressed in esophageal cancer cells. Here we investigated the function of TRAF6 in the esophageal cancer cell line EC109. siRNA targeting TRAF6 was introduced into EC109 cells and TRAF6 mRNA and protein levels were subsequently examined via RT-PCR and western blotting. Rates of apoptosis and cell proliferation were also measured using flow cytometry, ethynyl deoxyuridine (EdU), and CCK-8 (Cell Counting Kit-8) assays. The real-time PCR array was applied to profile the expression of TRAF6 related genes. TRAF6-siRNA reduced TRAF6 mRNA and protein expressions. NF-κB p65 protein expression was decreased in TRAF6-targeting siRNA-transfected cells compared to cells of the negative control. TRAF6-siRNA also significantly inhibited proliferation and enhanced apoptosis of EC109 cells. These studies suggested that TRAF6 was required for NF-κB activation in EC109 cells and it may be a good molecular target for suppressing the survival and proliferation of esophageal cancer cells.

Alternative Splicing of Pre-mRNA in the Control of Immune Activity.

The human immune response is a complex process that responds to numerous exogenous antigens in preventing infection by microorganisms, as well as to endogenous components in the surveillance of tumors and autoimmune diseases, and a great number of molecules are necessary to carry the functional complexity of immune activity. Alternative splicing of pre-mRNA plays an important role in immune cell development and regulation of immune activity through yielding diverse transcriptional isoforms to supplement the function of limited genes associated with the immune reaction. In addition, multiple factors have been identified as being involved in the control of alternative splicing at the cis, trans, or co-transcriptional level, and the aberrant splicing of RNA leads to the abnormal modulation of immune activity in infections, immune diseases, and tumors. In this review, we summarize the recent discoveries on the generation of immune-associated alternative splice variants, clinical disorders, and possible regulatory mechanisms. We also discuss the immune responses to the neoantigens produced by alternative splicing, and finally, we issue some alternative splicing and immunity correlated questions based on our knowledge.

Natural Antisense Transcript PEBP1P3 Regulates the RNA Expression, DNA Methylation and Histone Modification of CD45 Gene.

The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.

Bioinformatic analysis of the human DHRS4 gene cluster and a proposed mechanism for its transcriptional regulation.

BACKGROUND: The human DHRS4 gene cluster consists of three genes, DHRS4, DHRS4L2 and DHRS4L1. Among them, DHRS4 encodes NADP(H)-dependent retinol dehydrogenase/reductase. In a previous study, we investigated the alternative splicing of DHRS4 and DHRS4L2. DHRS4L1 was added to the gene cluster recently, but little is known about its structure and expression. To reveal the regulatory mechanism of the DHRS4 gene cluster expression, we studied the structure and transcription of DHRS4L1 in the context of the transcriptional behaviors of the human DHRS4 gene cluster. Based on the results of bioinformatics analysis, we propose a possible mechanism for the transcriptional regulation of the human DHRS4 gene cluster. RESULTS: The homologous comparison analysis suggests that DHRS4, DHRS4L2 and DHRS4L1 are three homologous genes in human. DHRS4L1 and DHRS4L2 are paralogues of DHRS4, and DHRS4L2 is the most recent member of the DHRS4 gene cluster. In the minus strand of the human DHRS4 gene cluster, a gene transcribed in an antisense direction was found containing a 5' sequence overlapping the region of exon 1 and promoter of DHRS4. By cloning the full length of RNA variants through 5'RACE and 3'RACE, we identified two transcription start sites, within exon a2 and exon 1, of this newly named gene DHRS4L1 using neuroblastoma cell line BE(2)-M17. Analysis of exon composition in the transcripts of DHRS4 gene cluster revealed that exon 1 was absent in all the transcripts initiated from exon a1 of DHRS4L2 and exon a2 of DHRS4L1. CONCLUSIONS: Alternatively spliced RNA variants are prevalent in the human DHRS4 gene cluster. Based on the analysis of gene transcripts and bioinformatic prediction, we propose here that antisense transcription may be involved in the transcriptional initiation regulation of DHRS4 and in the posttranscriptional splicing of DHRS4L2 and DRHS4L1 for the homologous identity of DHRS4 gene cluster. Beside the alternative transcriptional start sites, the antisense RNA is novel possible factor serving to remove exon 1 from the transcripts initiated from exon a1 and exon a2.

In vivo anti-tumor effect of hybrid vaccine of dendritic cells and esophageal carcinoma cells on esophageal carcinoma cell line 109 in mice with severe combined immune deficiency.

AIM: To develop a fusion vaccine of esophageal carcinoma cells and dendritic cells (DC) and observe its protective and therapeutic effect against esophageal carcinoma cell line 109 (EC109). METHODS: The fusion vaccine was produced by fusing traditional polyethyleneglycol (PEG), inducing cytokine, sorting CD34+ magnetic microbead marker and magnetic cell system (MACS). The liver, spleen and lung were pathologically tested after injection of the fusion vaccine. To study the therapeutic and protective effect of the fusion vaccine against tumor EC109, mice were divided immune group and therapeutic group. The immune group was divided into P, E, D and ED subgroups, immunized by phosphate buffered solution (PBS), inactivated EC109, DC and the fusion vaccine respectively, and attacked by EC109 cells. The tumor size, weight, latent period and mouse survival period were recorded and statistically analyzed. The therapeutic group was divided into four subgroups: P, inactivated EC109, D and ED subgroups, which were attacked by EC109 and then treated with PBS, inactivated EC109, DC, and EC109-DC respectively. Pathology and flow cytometry were also used to study the therapeutic effect of the fusion vaccine against EC109 cells. RESULTS: Flow cytometry showed that the expression of folate receptor (FR), EC109 (C), DCs (D) in human nasopharyngeal carcinoma cell line (HNE1) (B) was 78.21%, 89.50%, and 0.18%, respectively. The fusion cells (C) were highly expressed. No tumor was found in the spleen, lung and liver after injection of the fusion vaccine. Human IgG was tested in peripheral blood lymphocytes (PBL). In the immune group, the latent period was longer in EC109-DC subgroup than in other subgroups, while the tumor size and weight were also smaller than those in ED subgroup. In the therapeutic group, the tumor size and weight were smaller in ED subgroup than in P, inactivated EC109 and DC subgroups. CONCLUSION: Fusion cells are highly expressed not only in FR but also in CD80. The fusion vaccine has a distinctive protective effect against tumor EC109 and can inhibit the growth of tumor in mice, and its immune protection against tumor attack is more significant.

Enhancer RNA-driven looping enhances the transcription of the long noncoding RNA DHRS4-AS1, a controller of the DHRS4 gene cluster.

The human DHRS4 gene cluster consists of DHRS4 and two immediately downstream homologous genes, DHRS4L2 and DHRS4L1, generated by evolutionarily gene-duplication events. We previously demonstrated that a head-to-head natural antisense transcript (NAT) of DHRS4, denoted DHRS4-AS1, regulates all three genes of the DHRS4 gene cluster. However, it is puzzling that DHRS4L2 and DHRS4L1 did not evolve their own specific NATs to regulate themselves, as it seems both have retained sequences highly homologous to DHRS4-AS1. In a search of the DHRS4-AS1 region for nearby enhancers, we identified an enhancer located 13.8 kb downstream of the DHRS4-AS1 transcriptional start site. We further showed, by using a chromosome conformation capture (3C) assay, that this enhancer is capable of physically interacting with the DHRS4-AS1 promoter through chromosomal looping. The enhancer produced an eRNA, termed AS1eRNA, that enhanced DHRS4-AS1 transcription by mediating the spatial interactions of the enhancer and DHRS4-AS1 promoter in cooperation with RNA polymerase II and p300/CBP. Moreover, the distributions of activating acetyl-H3 and H3K4me3 modifications were found to be greater at the DHRS4-AS1 promoter than at the homologous duplicated regions. We propose that AS1eRNA-driven DNA looping and activating histone modifications promote the expression of DHRS4-AS1 to economically control the DHRS4 gene cluster.

Effects of dendritic cells from cord blood CD34+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice.

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in severe combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocarcinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11 d vs 7 d, P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

Oxidized low-density lipoprotein suppresses expression of prostaglandin E receptor subtype EP3 in human THP-1 macrophages.

EP3, one of four prostaglandin E2 (PGE2) receptors, is significantly lower in atherosclerotic plaques than in normal arteries and is localized predominantly in macrophages of the plaque shoulder region. However, mechanisms behind this EP3 expression pattern are still unknown. We investigated the underlying mechanism of EP3 expression in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages with oxidized low-density lipoprotein (oxLDL) treatment. We found that oxLDL decreased EP3 expression, in a dose-dependent manner, at both the mRNA and protein levels. Moreover, oxLDL inhibited nuclear factor-κB (NF-κB)-dependent transcription of the EP3 gene by the activation of peroxisome proliferator-activated receptor-γ (PPAR-γ). Finally, chromatin immunoprecipitation revealed decreased binding of NF-κB to the EP3 promoter with oxLDL and PPAR-γ agonist treatment. Our results show that oxLDL suppresses EP3 expression by activation of PPAR-γ and subsequent inhibition of NF-κB in macrophages. These results suggest that down-regulation of EP3 expression by oxLDL is associated with impairment of EP3-mediated anti-inflammatory effects, and that EP3 receptor activity may exert a beneficial effect on atherosclerosis.