Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2 V617F Mutation.

Precise quantification of the JAK2 V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2 V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2 V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.

Maternal exposure to particulate matter from duck houses restricts fetal growth due to inflammatory damage and oxidative stress.

The composition of particulate matter (PM) in poultry farms differs significantly from that of atmospheric PM as there is a higher concentration of microbes on farms. To assess the health effects of PM from poultry farms on pregnant animals, we collected PM from duck houses using a particulate sampler, processed it via centrifugation and vacuum concentration, and subsequently exposed the mice to airborne PM at 0.48 mg/m3 (i.e., low concentration group) and 1.92 mg/m3 (i.e., high concentration group) on the fifth day of pregnancy. After exposure until the twentieth day of pregnancy or spontaneous delivery, mice were euthanized for sampling. The effects of PM from duck houses on the pregnancy toxicity of mice were analyzed using histopathological analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction (qPCR). The results showed that exposure to PM had adverse effects on pregnant mice that reduced their feed intake in both groups. Microscopic lesions were observed in the lungs and placentas of pregnant mice, and the lesions worsened with increased PM concentrations, as shown by alveolar wall thickening, the infiltration of inflammatory cells in pulmonary interstitium, congestion, edema, and cellular degeneration of placenta. In pregnant mice in the high concentration group, exposure to PM significantly increased the expression of inflammatory cytokines in the lungs and placentas, caused oxidative stress, and decreased estrogen level in the blood. Exposure to PM also resulted in the reduced litter sizes of pregnant mice and shorter body and tail lengths in the fetuses delivered. Beyond that, exposure to PM significantly downregulated the levels of antioxidant factor superoxide dismutase and neurotrophic factor Ngf in the brains of fetuses. Collectively, exposure to a high concentration of PM by inhalation among pregnant mice caused significant pregnancy toxicity that led to abnormal fetal development due to inflammatory damage and oxidative stress. These findings established a foundation for future studies on the underlying mechanisms of pregnancy toxicity induced by exposure to PM.

Studies on the airborne bacterial communities and antimicrobial resistance genes in duck houses based on metagenome and PCR analysis.

The threat of antimicrobial resistance (AMR) is on the rise globally, especially with the development of animal husbandry and the increased demand for antibiotics. Livestock and poultry farms, as key sites for prevalence of antibiotic-resistant bacteria (ARB), can spread antimicrobial resistance genes (ARGs) through microbial aerosols and affect public health. In this study, total suspended particulate matter (TSP) and airborne culturable microorganisms were collected from duck houses in Tai'an, Shandong Province, and the bacterial communities and airborne ARGs were analyzed using metagenomics and PCR methods. The results showed that the bacterial communities in the air of duck houses were mainly Actinobacteria, Firmicutes, Proteobactria, Chlamydia, and Bcateroidetes at the phylum level. At the genus level, the air was dominated by Corynebacterium, Jeotgalicoccus, Staphylococcus, Brevibacterium, and Megacoccus, and contained some pathogenic bacteria such as Staphylococcus aureus, Corynebacterium diphtheriae, Klebsiella oxytoca, Acinetobacter baumannii, and Pseudomonas aeruginosa, which were also potential hosts for ARGs. The airborne ARGs were mainly macrolides (10.97%), penicillins (10.73%), cephalosporins (8.91%), streptozotocin (8.91%), and aminoglycosides (8.02%). PCR detected 27 ARGs in airborne culturable microorganisms, and comparative analysis between PCR and the metagenomic data revealed that a total of 9 ARGs were found to the same, including macrolides ErmA, ErmF, tetracyclines tetG, tetX, methicarbamazepines dfrA12, dfrA15, aminoglycosides APH3-VI, ANT2-Ⅰ, and sulfonamides sul2. Moreover, inhalation exposure modeling showed that the workers in duck houses inhaled higher concentrations of ARB, human pathogenic bacteria (HPB) and human pathogenic antibiotic-resistant bacteria (HPARB) than hospital workers. These results provide new insights into airborne microorganisms and ARGs in animal farms and lay the foundation for further study.

Methods, bioinformatics tools and databases in ecDNA research: An overview.

Extrachromosomal DNA (ecDNA), derived from chromosomes, is a cancer-specific circular DNA molecule. EcDNA drives tumor initiation and progression, which is associated with poor clinical outcomes and drug resistance in a wide range of cancers. Although ecDNA was first discovered in 1965, tremendous technological revolutions in recent years have provided crucial new insights into its key biological functions and regulatory mechanisms. Here, we provide a thorough overview of the methods, bioinformatics tools, and database resources used in ecDNA research, mainly focusing on their performance, strengths, and limitations. This study can provide important reference for selecting the most appropriate method in ecDNA research. Furthermore, we offer suggestions for the current bioinformatics analysis of ecDNA and provide an outlook to the future research.

M1 polarization of chicken macrophage HD11 can be activated by duck Tembusu virus via MyD88-NF-κB-mediated signaling pathway.

Duck Tembusu virus (DTMUV) has caused significant economic losses to the global duck industry since its outbreak in 2010. The macrophages act as the key immune cell, and its polarization in different functional states is very important for host's immune responses and microbial infections. Avian macrophages are the main target cells of DTMUV, its polarization induced by DTMUV and the underlying mechanisms were explored in this study. Through quantitative real-time PCR, nitrite assay, and flow cytometry analysis, we found that DTMUV caused severe inflammatory responses in chicken macrophage line HD11 by reprogramming the expression of M1- and M2-associated genes, leading to the polarization of HD11 macrophage to M1-type. In term of mechanism, transcriptomics was performed to analyze the M1-type polarization triggered by DTMUV, it was found that most differential genes were implicated in biological processes, and DTMUV infection significantly activated innate immune signaling pathways, including cytokine-cytokine receptor interaction, MAPK signaling pathway. Moreover, transcription factors NF-κB and AP1 also be activated after viral infection. However, further validation analysis by inhibitors and siRNAs of NF-κB and AP1 showed that NF-κB molecule was essential for DTMUV-induced M1 polarization in HD11 cell, but not AP1. Additionally, the inhibiting assays targeting MyD88 and TRIF molecules were conducted to determine their effect on NF-κB and M1-associated genes upregulated by DTMUV. The results showed that although the inhibition of both MyD88 and TRIF significantly downregulated the mRNA level of NF-κB, but the expression of M1-associated genes such as CD86 was lower in MyD88 inhibition group than in the other group, indicating that the role of MyD88 in mediating M1 polarization induced by DTMUV was more important. Overall, these results demonstrated that DTMUV infection induces M1-type polarization in chicken macrophage HD11 through MyD88-NF-κB signaling pathways. This finding will lay the foundation for further study the pathogenesis of DTMUV, and provide new insights into the prevention and control of this disease.

Efficacy, toxicity and prognostic factors of pyrotinib­involved neoadjuvant therapy in HER2­positive breast cancer: A retrospective study.

Pyrotinib is a novel irreversible tyrosine kinase inhibitor targeting the human epidermal growth factor receptor (HER), whose efficacy in treating metastatic HER2-positive (HER2+) breast cancer has been confirmed. The present study aimed to explore the efficacy, safety and prognostic factors of pyrogenic-involved neoadjuvant therapy in patients with HER2+ breast cancer. A total of 49 patients with HER2+ breast cancer who received pyrotinib-neoadjuvant therapy were recruited. All patients received pyrotinib plus chemotherapy with or without trastuzumab neoadjuvant treatment for six cycles (21 days/cycle). Concerning the clinical response, 4 (8.2%), 36 (73.4%) and 9 (18.4%) patients achieved complete response, partial response and stable disease after 6-cycle pyrotinib-neoadjuvant treatment, respectively; the objective response rate and disease control rate reached 81.6 and 100.0%, respectively. Concerning the pathological response, 23 (46.9%), 12 (24.5%), 12 (24.5%) and 2 (4.1%) patients were evaluated as Miller-Payne grade 5, 4, 3 and 2, respectively. In addition, 23 (46.9%) patients achieved pathological complete response (pCR) in the breast tissue, 40 (81.6%) patients achieved pCR in lymph nodes, while 22 (44.9%) patients obtained total pCR (tpCR). Further multivariate logistic regression analysis demonstrated that pyrotinib plus trastuzumab and chemotherapy (vs. pyrotinib plus chemotherapy) was independently correlated with increased tpCR (P=0.048). The most frequent adverse events included diarrhea (81.6%), anemia (69.4%), nausea and vomiting (63.3%), and fatigue (51.0%). The majority of the adverse events were mild and controllable. In conclusion, pyrotinib-neoadjuvant therapy presented optimal efficacy and mild toxicity in patients with HER2+ breast cancer, whose efficacy was affected by the combination treatment with trastuzumab.

Epidemiological investigation and drug resistance characteristics of Riemerella anatipestifer strains from large-scale duck farms in Shandong Province, China from March 2020 to March 2022.

Infectious serositis is a common disease caused by Riemerella anatipestifer (R. anatipestifer) in ducks, characterized by respiratory distress, septicemia, and neurological symptoms. In this study, 1,020 samples (brain and liver) were collected from ducks with suspected R. anatipestifer infection from March 2020 to March 2022 in Shandong Province, of which 171 R. anatipestifer strains were identified by PCR and isolation culture. The serotype of all strains was analyzed, and 74 strains were subjected to drug sensitivity tests and drug resistance genes detection. The results showed that the overall prevalence rate of R. anatipestifer in Shandong Province was 16.7% (171/1,020), with most strains coming from brain samples of ducklings under 3-mo old collected from September to December each year. Histopathological examination showed that heart vessels of the diseased duck were highly dilated and filled with red blood cells, with obvious fibrin exudates outside the pericardium, and fatty degeneration of liver cells. There were 45 strains of serotype 1, 45 strains of serotype 2, 2 strains of serotype 4, 33 strains of serotype 6, 44 strains of serotype 7, and 2 strains of serotype 10. The minimum inhibitory concentration (MIC) of 10 common antibiotics against 74 representative strains was determined by the agar dilution method. It was found that 74 strains had the most severe resistance to gentamicin (77%) and fully susceptible to ceftriaxone, but the 81.1% isolated strains were multidrug resistant. Resistance genes testing of 74 R. anatipestifers showed that tetracycline resistance gene tet X had the highest detection rate of 95.9%, followed by macrolide resistance gene ermF with 77%, and the rate of ß-lactam resistance gene blaTEM is the lowest (10.8%). The animal experiment of 4 R. anatipestifer strains with different serotypes showed that they had strong pathogenicity to 7-day-old ducklings, which could cause nervous symptoms, and the mortality rate was 58% to 70%. The autopsy showed obvious pathological changes. These findings of this study on R. anatipestifer will help us to understand the latest prevalence, drug resistance characteristics, and pathogenicity of R. anatipestifer in Shandong, China, and provide a scientific guide for the treatment and control of the disease.

Retrospective Investigation and Genetic Variation Analysis of Chicken Infectious Anemia in Shandong Province, 2020-2022.

Chicken infectious anemia (CIA) is a vertical transmission infectious chicken disease caused by the chicken infectious anemia virus (CAV). The disease can induce stunting and immunosuppression in chicks by infecting bone marrow-derived stem cells, causing huge economic losses for the poultry industry. To determine the prevalence of CIA in Shandong Province, China, 854 suspected CIA samples were collected and analyzed in 13 cities in Shandong from 2020 to 2022. The PCR results showed that a total of 115 CAV were isolated. The CAV-positive rates were 17.21% (26/151) in 2020, 12.23% (35/286) in 2021, and 12.94% (54/417) in 2022, with severe mixed infections. Among them, CAV and fowl adenovirus (FAdV) were the most common, accounting for 40.86%. VP1 gene homology analysis showed that isolated strains shared 96.1-100% homology with the previously reported CAV strains. Genetic variation analysis showed that most of the isolated CAV strains were located in genotype A. These results indicate that CIA infection in Shandong chickens in recent years has been prevalent and mixed infections are common, but there were no significant genetic variations. Our results extend the understanding of the prevalence and genetic evolution of CIA in Shandong Province. They will offer new references for further study of the epidemiology and virus variation and the prevention and control of this disease.

Genomic Characteristics and Phylogenetic Analyses of a Multiple Drug-Resistant Klebsiella pneumoniae Harboring Plasmid-Mediated MCR-1 Isolated from Tai'an City, China.

Klebsiella pneumoniae is a clinically common opportunistic pathogen that causes pneumonia and upper respiratory tract infection in humans as well as community-and hospital-acquired infections, posing significant threats to public health. Moreover, the insertion of a plasmid carrying the mobile colistin resistance (MCR) genes brings obstacles to the clinical treatment of K. pneumoniae infection. In this study, a strain of colistin-resistant K. pneumoniae (CRKP) was isolated from sputum samples of a patient who was admitted to a tertiary hospital in Tai'an city, China, and tested for drug sensitivity. The results showed that KPTA-2108 was multidrug-resistant (MDR), being resistant to 21 of 26 selected antibiotics, such as cefazolin, amikacin, tigecycline and colistin but sensitive to carbapenems via antibiotic resistance assays. The chromosome and plasmid sequences of the isolated strain KPTA-2108 were obtained using whole-genome sequencing technology and then were analyzed deeply using bioinformatics methods. The whole-genome sequencing analysis showed that the length of KPTA-2108 was 5,306,347 bp and carried four plasmids, pMJ4-1, pMJ4-2, pMJ4-3, and pMJ4-4-MCR. The plasmid pMJ4-4-MCR contained 30,124 bp and was found to be an IncX4 type. It was the smallest plasmid in the KPTA-2108 strain and carried only one resistance gene MCR-1. Successful conjugation tests demonstrated that pMJ4-4-MCR carrying MCR-1 could be horizontally transmitted through conjugation between bacteria. In conclusion, the acquisition and genome-wide characterization of a clinical MDR strain of CRKP may provide a scientific basis for the treatment of K. pneumoniae infection and epidemiological data for the surveillance of CRKP.

eccDNA Atlas: a comprehensive resource of eccDNA catalog.

Extrachromosomal circular DNA (eccDNA) represents a large category of non-mitochondrial and non-plasmid circular extrachromosomal DNA, playing an indispensable role in various aspects such as tumorigenesis, immune responses. However, the information of characteristics and functions about eccDNA is fragmented, hiding behind abundant literatures and massive whole-genome sequencing (WGS) data, which has not been sufficiently used for the identification of eccDNAs. Therefore, establishing an integrated repository portal is essential for identifying and analyzing eccDNAs. Here, we developed eccDNA Atlas (http://lcbb.swjtu.edu.cn/eccDNAatlas), a user-friendly database of eccDNAs that aims to provide a high-quality and integrated resource for browsing, searching and analyzing eccDNAs from multiple species. eccDNA Atlas currently contains 629 987 eccDNAs and 8221 ecDNAs manually curated from literatures and 1105 ecDNAs predicted by AmpliconArchitect based on WGS data involved in 66 diseases, 57 tissues and 319 cell lines. The content of each eccDNA entry includes multiple aspects such as sequence, disease, function, characteristic, validation strategies. Furthermore, abundant annotations and analyzing utilities were provided to explore existing eccDNAs in eccDNA Atlas or user-defined eccDNAs including oncogenes, typical enhancers, super enhancers, CTCF-binding sites, SNPs, chromatin accessibility, eQTLs, gene expression, survival and genome visualization. Overall, eccDNA Atlas provides an integrated eccDNA data warehouse and serves as an important tool for future research.

Primary hepatic paraganglioma mimicking hepatocellular carcinoma: a case report.

Background: Primary hepatic paraganglioma (HPGL) originates from sympathetic nervous tissue in the liver. It is one of an exceedingly rare kind of sympathetic paragangliomas. The radiological features and clinical characters of HPGL can be easily confused with hepatocellular carcinoma (HCC). We present a case of HCC that was preoperatively diagnosed as hepatic paraganglioma, however, was pathologically verified as hepatic paraganglioma after surgery. Case Description: The present case reported a 47-year-old female with a very rare HPGL without any clinical symptoms, except for hyper menorrhagia and paroxysmal hypertension. The Spiegelman lobe of the liver underwent hepatic magnetic resonance imaging, which revealed a 3.2×3.8 cm mass, with uneven arterial phase wash-in and rapid portal and delayed phase wash-out. According to the imaging results, the patient was first diagnosed with hepatocellular carcinoma, and a radical hepatectomy was performed. However, the blood pressure of the patient displayed dramatic changes when the tumor was stimulated in operation. There were no substantial abnormalities found in the bilateral renal and adrenal glands. Therefore, we presumed that the tumor was related to functional pheochromocytoma. The tumor tissue was shown to be positive for chromogranin A, synaptophysin, CD56, and vimentin by immunohistochemical analysis. As a result, the patient was diagnosed with HPGL after this pathologic evaluation. Conclusions: There are several similarities between HPGL and HCC. For the treatment of hepatic paraganglioma, surgical excision is the recommended practice. Although the majority of paragangliomas are benign, long-term monitoring is required to differentiate benign from malignant paragangliomas.

Tauroursodeoxycholic acid inhibits TGF-ß1-induced renal fibrosis markers in cultured renal mesangial cells by regulating endoplasmic reticulum stress.

Chronic kidney disease (CKD) has a worldwide prevalence of higher than 10% with an increasing mortality rate. As it involves the deterioration of renal function, it represents a serious risk to human health and, if left untreated, significantly lowers the quality of the patient's life. CKD is characterized by renal fibrosis. Studies have shown that transforming growth factor ß1 (TGF-ß1), a key driving factor of renal fibrosis, is closely related to the activation of renal fibrosis pathways such as endoplasmic reticulum stress (ERS). Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid derivative, can effectively inhibit endogenous ERS. Here, we explored the effects and actions of TUDCA on renal fibrosis by establishing a renal mesangial cell (RMC) model. The RMC was stimulated with TGF-ß1, and PCR and western blotting were used to detect the expression of ERS-related chaperone proteins and fibrotic indicators. The expression of glucose-regulated protein 78 (GRP78) was silenced in RMC cells to investigate the role of GRP78 in renal fibrosis. Finally, PCR and western blotting were used to detect the effects of TUDCA on the expression of GRP78, C/EBP homologous protein (CHOP), α-smooth muscle actin (α-SMA), and fibronectin (FN) in the TGF-ß1-stimulated RMCs. The results showed that TUDCA significantly downregulated TGF-ß1-induced levels of GRP78, CHOP, α-SMA and FN in RMCs. In addition, downregulation of GRP78 inhibited the expression of FN and α-SMA in the RMCs. In conclusion, downregulation of GRP78 and CHOP expression is one of the mechanisms by which TUDCA inhibits TGF-ß1-induced renal mesangial cell fibrosis.

Non-Structural Protein 3 of Duck Tembusu Virus Induces Autophagy via the ERK and PI3K-AKT-mTOR Signaling Pathways.

Despite autophagy's pivotal role in the replication of viruses such as duck Tembusu virus (DTMUV), which has caused massive economic losses to the poultry industry in the world, the specific relationships between DTMUV and cellular autophagy remain largely unknown. In response, we investigated the interactions between autophagy and DTMUV, the effects of the structural and non-structural proteins of DTMUV on autophagy, and the autophagy-related signaling pathways induced by DTMUV. Among the results, DTMUV increased the autophagy flux in duck embryo fibroblasts (DEF) and BHK-21 cells, while autophagy facilitated viral replication. After we pharmacologically induced autophagy with rapamycin (RAPA), the replication of DTMUV increased by 15.23-fold compared with the control group of DEF cells. To identify which DTMUV protein primarily induced autophagy, all three structural proteins and seven non-structural proteins of DTMUV were transfected into cells, and the results showed that non-structural protein 3 (NS3) induced significant autophagy in DEF cells. By means of Western blot, immunofluorescence, and transmission electron microscopy, we confirmed that NS3 protein could significantly induce autophagy and autophagy flux. Furthermore, we showed that NS3 induced autophagy in DEF cells through extracellular signal-regulated kinase 2 (ERK2) and phosphatidylinositol-3-kinase (PI3K)/AKT and the mammalian target of rapamycin (mTOR) signaling pathways using specific inhibitors and RNA interference assays. Finally, autophagy induced by NS3 promoted DTMUV replication. These results provide novel insight into the relationship between DTMUV and autophagy, broadening the current understanding of the molecular pathogenesis of DTMUV.

Tfcancer: a manually curated database of transcription factors associated with human cancers.

SUMMARY: Transcription factors (TFs) are critical regulation elements and its dysregulation can lead to a variety of cancers. However, currently, there are no such online resources for large-scale collection, storage and analysis of TF-cancer associations in those cancers. To fill this gap, we present a database called TFcancer (http://lcbb.swjtu.edu.cn/tfcancer/), which contains 3136 experimentally supported associations between 364 TFs and 33 TCGA cancers by manually curating more than 1800 literature. TFcancer mainly concentrates on four aspects: TF expression, molecular alteration, regulatory relationships between TFs and target genes, and biological processes and signaling pathways of TFs in cancers. TFcancer not only provides a user-friendly interface for browsing and searching but also allows flexible data downloading and user data submitting. It is believed that TFcancer is a helpful and valuable resource for researchers who seek to understand the functions and molecular mechanisms of TFs involved in human cancers. AVAILABILITY AND IMPLEMENTATION: The TFcancer are freely available at http://lcbb.swjtu.edu.cn/tfcancer/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification and characteristic analysis of enhancers across 13 major cancer types.

Enhancers are often mutated and dysregulated in various diseases such as cancer. By integrating the function annotation of the mammalian genome (FANTOM) enhancers expression profiles and RNA-seq data from The Cancer Genome Atlas (TCGA) of 13 cancers and their corresponding para-cancerous tissues, we systematically identified a total of 4702 significantly differentially expressed (DE) enhancers. Furthermore, a total of 1036 DE genes regulated by DE enhancers were identified. It was found that in these 13 cancers, most (61.13%) enhancers were ubiquitously expressed, whereas DE enhancers were more likely to be tissue-specific expressed, and the DE genes regulated by DE enhancers were significantly enriched in cancer-related pathways. Finally, it was manifested that 74 single nucleotide polymorphisms (SNPs) were located in 37 DE enhancers, and these SNPs affected the gain and loss of functional transcription factor binding sites of 758 transcription factors, which were shown to be highly correlated with tumorigenesis and development.

EnhFFL: A database of enhancer mediated feed-forward loops for human and mouse.

Feed-forward loops (FFLs) are thought to be one of the most common and important classes of transcriptional network motifs involved in various diseases. Enhancers are cis-regulatory elements that positively regulate protein-coding genes or microRNAs (miRNAs) by recruiting DNA-binding transcription factors (TFs). However, a comprehensive resource to identify, store, and analyze the FFLs of typical enhancer and super-enhancer FFLs is not currently available. Here, we present EnhFFL, an online database to provide a data resource for users to browse and search typical enhancer and super-enhancer FFLs. The current database covers 46 280/7000 TF-enhancer-miRNA FFLs, 9997/236 enhancer-miRNA-gene FFLs, 3 561 164/3 193 182 TF-enhancer-gene FFLs, and 1259/235 TF-enhancer feed-back loops (FBLs) across 91 tissues/cell lines of human and mouse, respectively. Users can browse loops by selecting species, types of tissue/cell line, and types of FFLs. EnhFFL supports searching elements including name/ID, genomic location, and the conservation of miRNA target genes. We also developed tools for users to screen customized FFLs using the threshold of q value as well as the confidence score of miRNA target genes. Disease and functional enrichment analysis showed that master miRNAs that are widely engaged in FFLs including TF-enhancer-miRNAs and enhancer-miRNA-genes are significantly involved in tumorigenesis. Database URL:http://lcbb.swjtu.edu.cn/EnhFFL/.

Identification and Analysis of p53-Regulated Enhancers in Hepatic Carcinoma.

Enhancers can act as cis-regulatory elements to control transcriptional regulation by recruiting transcription factors (TFs) in a distance and orientation-independent manner. However, it is still unclear how p53 participates in the enhancer network as TF in hepatic carcinoma under the condition of DNA damage. A total of 14,286 active enhancers were identified through the integration of stable and unstable enhancer RNAs (eRNAs) captured by CAGE and GRO-seq, respectively. Furthermore, 218 p53-bound enhancers (Enhp53) were identified by analyzing p53 ChIP-seq in HepG2 cells after DNA damage. The results showed that the enhancer expression and histone markers of enhancers (H3K4me1, H3K4me2, H3K4me3, H3K9ac, and H3K27ac) revealed significantly higher level on Enhp53 than Enhno-p53 which suggested that p53 participated in regulating enhancer activity and chromatin structure. By analyzing 124 TFs ChIP-seq from ENCODE, 93 TFs were found significantly enriched on Enhp53 such as GATA4, YY1, and CTCF, indicating p53 may co-regulate enhancers with TFs participation. Moreover, significantly differentially expressed 438 miRNAs and 1,264 mRNAs were identified by analyzing small RNA-seq and RNA-seq, and 26 Enhp53-miRNAs and 145 Enhp53-mRNA interactions were identified by the integration of 3D genome data and genomic distance. The functional enrichment analysis showed that these miRNA targets and mRNAs were significantly involved in tumor biological processes and signaling pathways such as DNA replication, p53 signaling pathway, hepatitis B, focal adhesion, etc. The above results indicated that p53 participated in regulating enhancer network in hepatic carcinoma and Enhp53 exhibited significantly different characteristics with Enhno-p53.

Genome-Wide Identification and Analysis of Enhancer-Regulated microRNAs Across 31 Human Cancers.

Enhancers are cis-regulatory DNA elements that positively regulate the transcription of target genes in a tissue-specific manner, and dysregulation of target genes could lead to various diseases, such as cancer. Recent studies have shown that enhancers can regulate microRNAs (miRNAs) and participate in their biological synthesis. However, the network of enhancer-regulated miRNAs across multiple cancers is still unclear. Here, a total of 2,418 proximal enhancer-miRNA interactions and 1,280 distal enhancer-miRNA interactions were identified through the integration of genomic distance, co-expression, and 3D genome data in 31 cancers. The results showed that both proximal and distal interactions exhibited a significant cancer type-specific feature trend at the tissue level rather than at the single-cell level, and there was a noteworthy positive correlation between the expression of the miRNA and the number of enhancers regulating the same miRNA in most cancers. Furthermore, we found that there was a high correlation between the formation of enhancer-miRNA pairs and the expression of enhancer RNAs (eRNAs) whether in distal or proximal regulation. The characteristics analysis showed that miRes (enhancers that regulated miRNAs) and non-miRes presented significant differences in sequence conservation, guanine-cytosine (GC) content, and histone modification signatures. Notably, GC content, H3K4me1, and H3K36me3 were present differently between distal and proximal regulation, suggesting that they might participate in chromosome looping of enhancer-miRNA interactions. Finally, we introduced a case study, enhancer: chr1:1186391-1186507 ∼ miR-200a was highly relevant to the survival of thyroid cancer patients and a cis-eQTL SNP on the enhancer affected the expression of the TNFRSF18 gene as a tumor suppressor.

Down-regulation expression of TGFB2-AS1 inhibits the proliferation, migration, invasion and induces apoptosis in HepG2 cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of cancer mortality and without effective prognosis. Previous study has been confirmed that the abnormal expression of long non-coding RNAs (lncRNAs) TGFB2-AS1 was involved in tumorigenesis. However, the biological functions of TGFB2-AS1 in hepatocellular carcinoma (HCC) remain largely unclear. OBJECTIVE: We comprehensively assess the clinical significance of TGFB2-AS1 and investigate the biological functions of TGFB2-AS1 on HCC HepG2 cells. METHODS: We firstly confirmed the expression of TGFB2-AS1 between tumor and normal tissues using public available transcriptome data. We analyzed the clinical significance of TGFB2-AS1 using the TCGA HCC datasets. The biological functions of TGFB2-AS1 on HCC HepG2 cells were explored by multiple in vitro assays. RESULTS: We found that TGFB2-AS1 was remarkably increased in HCC tissues (P = 0.00148) and exhibited a potential predictive marker for HCC, with an area under curve (AUC) of 0.708 (P = 0.0034) using the fifty pairs of matched HCC tissues of TCGA. Besides, higher expression of TGFB2-AS1 in HCC tissues was identified as being positively associated with advanced tumor (P = 0.012) and disease stage (P = 0.009) in 355 HCC cases using independent sample nonparametric test. Downregulation of TGFB2-AS1 expression significantly restrained proliferation (P < 0.01) and impaired colony formation (P < 0.05). Furthermore, TGFB2-AS1 depletion remarkably promoted the apoptosis of HepG2 cells (P < 0.05) and inhibited migration and invasion (P < 0.01). CONCLUSION: Taken together, these findings suggested that TGFB2-AS1 might serve as a potential therapeutic target for HCC.

EnhancerDB: a resource of transcriptional regulation in the context of enhancers.

Enhancers can act as cis-regulatory elements to control transcriptional regulation by recruiting DNA-binding transcription factors (TFs) in a tissue-specific manner. Recent studies show that enhancers regulate not only protein-coding genes but also microRNAs (miRNAs), and mutations within the TF binding sites (TFBSs) located on enhancers will cause a variety of diseases such as cancer. However, a comprehensive resource to integrate these regulation elements for revealing transcriptional regulations in the context of enhancers is not currently available. Here, we introduce EnhancerDB, a web-accessible database to provide a resource to browse and search regulatory relationships identified in this study, including 131 054 581 TF-enhancer, 17 059 enhancer-miRNAs, 318 993 enhancer-genes, 4 639 558 TF-miRNAs, 1 059 695 TF-genes, 11 439 394 enhancer-single-nucleotide polymorphisms (SNPs) and 23 334 genes associated with expression quantitative trait loci (eQTL) SNP and expression profile of TF/gene/miRNA across multiple human tissues/cell lines. We also developed a tool that further allows users to define tissue-specific enhancers by setting the threshold score of tissue specificity of enhancers. In addition, links to external resources are also available at EnhancerDB.