Gene Network Analysis Research Articles

Identification of tRNA-derived small RNAs and their potential roles in porcine skeletal muscle with intrauterine growth restriction.

Intrauterine growth restriction (IUGR) in humans often manifests as poor growth and delayed intellectual development, whereas in domestic animals it results in increased mortality. As a novel epigenetic regulatory molecule, tRNA-derived small RNAs (tsRNAs) have been reported to be involved in many biological processes. In this study, pigs (35d) were used as a model to characterize tsRNAs by sequencing in normal and IUGR porcine skeletal muscle. A total of 586 tsRNAs were identified, of which 103 were specifically expressed in normal-size pigs and 38 were specifically expressed in IUGR pigs. The tsRNAs formed by splicing before the 5' end anti codon of mature tRNA (tRF-5c) accounted for over 90% of tsRNAs, which were significantly enriched in IUGR pigs than in normal-size pigs. Enriched pathways of differentially expressed tsRNAs target genes mainly included metabolic pathways, Rap1 signaling pathway, endocytosis, mTOR signaling pathway, and AMPK signaling pathway. Regulatory network analysis of target genes revealed that IGF1 was one of the most important molecules of regulatory nodes in IUGR and normal porcine skeletal muscle. In addition, IGF1 was found to be one of the target genes of tRF-Glu-TTC-047, which is a highly expressed tsRNA in IUGR pigs. The findings described herein uncover the role of tsRNAs in IUGR porcine skeletal muscle development, thus providing insights into the prevention and treatment of IUGR in mammals.

Analysis of Differentially Expressed MicroRNAs in OVA-induced Airway Remodeling Model Mice.

MicroRNAs (miRNAs) can participate in airway remodeling by regulating immunemolecule expression. Here, we aimed to identify the differential miRNAs involved in airway remodeling. Airway remodeling was induced by ovalbumin in female BALB/C mice. The differentially expressed miRNAs were screened with microarray. GO (Gene Ontology) and KEGG enrichment analysis was performed. The miRNA target gene network and miRNA target pathway network were constructed. Verification with real-time PCR and Western blot was performed. We identified 63 differentially expressed miRNAs (50 up-regulated and 13 down-regulated) in the lungs of ovalbumin-induced airway remodeling mice. Real-time PCR confirmed that 3 miRNAs (mmu-miR-1931, mmu-miR-712-5p, and mmu-miR-770-5p) were significantly up-regulated, and 4 miRNAs (mmu-miR-128-3p, mmu-miR-182-5p, mmu-miR-130b-3p, and mmu-miR-20b-5p) were significantly down-regulated. The miRNA target gene network analysis identified key mRNAs in the airway remodeling, such as Tnrc6b (trinucleotide repeat containing adaptor 6B), Sesn3 (sestrin 3), Baz2a (bromodomain adjacent to zinc finger domain 2a), and Cux1 (cut like homeobox 1). The miRNA target pathway network showed that the signal pathways such as MAPK (mitogen-activated protein kinase), PI3K/Akt (phosphoinositide 3-Kinase/protein kinase B), p53 (protein 53), and mTOR (mammalian target of rapamycin) were closely related to airway remodeling in asthma. Collectively, differential miRNAs involved in airway remodeling (such as mmu-miR-1931, mmu-miR-712-5p, mmu-miR-770-5p, mmu-miR-128-3p mmu-miR-182-5p, and mmu-miR-130b-3p) as well as their target genes (such as Tnrc6b, Sesn3, Baz2a, and Cux1) and pathways (such as MAPK, PI3K/Akt, p53, mTOR pathways) have been identified. Our findings may help to further understand the pathogenesis of airway remodeling.

WIN Symposium 2022 - Abstract Poster Presentations.

WIN Symposium 2022 - Abstract Poster Presentations.

Unraveling the key driving factors involved in cometabolism enhanced aerobic degradation of tetracycline in wastewater

Cometabolism has shown great potential in increasing the engineering feasibility of microalgae-based biotechnologies for the aerobic treatment of antibiotics-polluted wastewaters. Yet, the underlying mechanisms involved in improved microalgal performance remain unknown. In this study, we incorporated transcriptomics, gene network analysis, and enzymatic activities with cometabolic pathways of tetracycline (TC) by Chlorella pyrenoidosa to identify the key driving factors. The results demonstrated that cometabolism constructed a metabolic enzymes-photosynthetic machinery to improve the electron transport chain and activities of catalytic enzymes, which resulted in subsequent 100% removal of TC. Coupling formation dynamics of the intermediates with roles of identified metabolic enzymes, degradation of TC can be induced by de/hydroxylation, de/hydrogenation, bond-cleavage, decarboxylation, and deamination. Evaluation of 18 antibiotics’ removal in reclaimed water showed cometabolism decreased the total concentrations of these antibiotics from 495.54 ng L−1 to 221.80 ng L−1. Our findings not only highlight the application potential of cometabolism in increasing engineering feasibility of microalgal degradation of antibiotics from wastewaters, but also provide the unique insights into unraveling the “black-box” of cometabolisms in aerobic biodegradation.

Time-Varying Gene Expression Network Analysis Reveals Conserved Transition States in Hematopoietic Differentiation between Human and Mouse.

(1) Background: analyses of gene networks can elucidate hematopoietic differentiation from single-cell gene expression data, but most algorithms generate only a single, static network. Because gene interactions change over time, it is biologically meaningful to examine time-varying structures and to capture dynamic, even transient states, and cell-cell relationships. (2) Methods: a transcriptomic atlas of hematopoietic stem and progenitor cells was used for network analysis. After pseudo-time ordering with Monocle 2, LOGGLE was used to infer time-varying networks and to explore changes of differentiation gene networks over time. A range of network analysis tools were used to examine properties and genes in the inferred networks. (3) Results: shared characteristics of attributes during the evolution of differentiation gene networks showed a “U” shape of network density over time for all three branches for human and mouse. Differentiation appeared as a continuous process, originating from stem cells, through a brief transition state marked by fewer gene interactions, before stabilizing in a progenitor state. Human and mouse shared hub genes in evolutionary networks. (4) Conclusions: the conservation of network dynamics in the hematopoietic systems of mouse and human was reflected by shared hub genes and network topological changes during differentiation.

Effect of Maternal Body Mass Index on the Transcriptomic Network of Human First-Trimester Chorionic Villi.

The relationship between fertility and maternal body weight is shaped like an inverted "U," meaning that fertility is negatively affected in overweight or underweight women. Timely and appropriate maternal-fetal interaction is a crucial part of successful pregnancy. However, it is not clear how body weight affects maternal-fetal interaction. Placental villi are the bridge for maternal-fetal interaction. Therefore, we collected villi from pregnant women with different body mass indexes (BMI), who voluntarily underwent induced abortion, to construct a molecular network via RNA-seq. Surprisingly, based on global and significant gene network analysis, we found that dysregulation of inflammatory reaction, cell adhesion, and immune response were the most significantly enriched pathways. We also conducted dynamic gene expression analysis with BMI as a variable, and identified several distinct clusters. Among them, cluster 9 showed an inverted "U" shape and genes in it were mainly enriched in chemical synaptic transmission and cell-cell adhesion via plasma-membrane adhesion molecules. Additionally, genes in the "U" shaped cluster (cluster 5) were enriched in regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains and negative regulation of immune response. We thus conclude that maternal body weight can affect maternal-fetal interaction through alterations or aberrant activation of inflammatory reaction and immune response. Regulating inflammatory reaction may be a potential therapeutic strategy to improve fertility of overweight and underweight people.

The spatial transcriptomic landscape of non-small cell lung cancer brain metastasis

Brain metastases (BrMs) are a common occurrence in lung cancer with a dismal outcome. To understand the mechanism of metastasis to inform prognosis and treatment, here we analyze primary and metastasized tumor specimens from 44 non-small cell lung cancer patients by spatial RNA sequencing, affording a whole transcriptome map of metastasis resolved with morphological markers for the tumor core, tumor immune microenvironment (TIME), and tumor brain microenvironment (TBME). Our data indicate that the tumor microenvironment (TME) in the brain, including the TIME and TBME, undergoes extensive remodeling to create an immunosuppressive and fibrogenic niche for the BrMs. Specifically, the brain TME is characterized with reduced antigen presentation and B/T cell function, increased neutrophils and M2-type macrophages, immature microglia, and reactive astrocytes. Differential gene expression and network analysis identify fibrosis and immune regulation as the major functional modules disrupted in both the lung and brain TME. Besides providing systems-level insights into the mechanism of lung cancer brain metastasis, our study uncovers potential prognostic biomarkers and suggests that therapeutic strategies should be tailored to the immune and fibrosis status of the BrMs.

Achieving tolerance modifies cancer susceptibility profiles in liver transplant recipients.

Long-term survival of transplant recipients is significantly impacted by malignancy. We aimed to determine whether calcineurin inhibitor (CNI)-treated recipients converted to and weaned off molecular target of rapamycin inhibitor (mTOR-I) therapy have favorable changes in their molecular profiles in regard to malignancy risk. We performed gene expression profiling from liver biopsy and blood (PBMC) specimens followed by network analysis of key dysregulated genes, associated diseases and disorders, molecular and cellular functions using IPA software. Twenty non-immune, non-viremic patients were included, and 8 of them achieved tolerance. Two comparisons were performed: (1) tolerance time point vs tacrolimus monotherapy and (2) tolerance time point vs sirolimus monotherapy. Upon achieving tolerance, IPA predicted significant activation of DNA damage response (p=5.40e-04) and inhibition of DNA replication (p=7.56e-03). Conversion from sirolimus to tolerance showed decrease in HCC (p=1.30e-02), hepatic steatosis (p=5.60e-02) and liver fibrosis (p=2.91e-02) associated genes. In conclusion, this longitudinal study of patients eventually achieving tolerance reveals an evolving molecular profile associated with decreased cancer risk and improved hepatic steatosis and liver fibrosis. This provides a biological rationale for attempting conversion to mTOR-I therapy and tolerance following liver transplantation particularly in patients at higher risk of cancer incidence and progression post-transplant.

Roles of Curcumin on Cognitive Impairment Induced by a Mixture of Heavy Metals.

We aimed to explore the molecular mechanisms of curcumin's protective action against heavy metal-related cognitive impairment (CI). In silico analysis, CTD, SwissADME, AutoDock Vina, Metascape, GeneMania, and MIENTURNET were key approaches. The server-predicted interactions (41.7%) and physical interactions (35.7%) were found to be the most important interactions in the gene network analysis. The most important pathways involved in curcumin's protective activity against heavy metals were categorized as "regulation of neuron apoptotic process" and "negative regulation of apoptotic signaling route". These pathways were also emphasized in the protein-protein interaction enrichment analysis. Curcumin was also well-positioned inside the CASP3 binding region. Three key miRNAs linked to CI, mixed heavy metals, and curcumin (hsa-miR-34a-5p, hsa-miR-24-3p, and hsa-miR-128-3p)were observed. These miRNAs were found to be related to the important pathways related to CI and involved in curcumin's protective activity against mixed heavy metals such as "apoptosis multiple species", "apoptosis", and "Alzheimer's disease". We also created and tested in silico sponges that inhibited these miRNAs. Curcumin's physicochemical characteristics and pharmacokinetics are consistent with its therapeutic benefits in CI, owing to its high gastrointestinal absorption and ability to cross the blood-brain barrier, and it is not a P-glycoprotein substrate. Our findings emphasize the protective effects of curcumin in CI caused by heavy metal mixtures and pave the way for molecular mechanisms involved in CI pathology.

Transcriptomic analysis of uterine receptivity in Guinea pigs

The mouse is a widely used animal model for studying human reproduction. However, the mice are eccentric implantation, and ovarian progesterone and estrogen are essential for implantation, which is different from human beings. Guinea pigs are unique from mice since, like humans, they are the interstitial implantation, and ovarian progesterone alone seems to be sufficient to allow for implantation. In this study, we firstly analyzed the gene expression profiles in the receptive uterus on day 5 of pregnancy compared to the pre-receptive uterus on day 3 of pregnancy in guinea pigs using the RNA-seq method. In total, 304 up-regulated and 475 down-regulated genes were identified. Gene Ontology and pathway enrichment analyses highlighted the importance of the inflammatory response in the receptive uterus. Through gene network analysis, we identified 24 hub genes, most of which were involved in regulating endometrial receptivity. In addition, we performed a cross-species comparison of differentially expressed genes associated with uterine receptivity. A total of 14 genes were shared among guinea pigs, humans, and mice. Furthermore, Fisher's exact test revealed that gene expression patterns of uterine receptivity were more similar between guinea pigs and humans than between mice and humans. Our study may contribute to increasing the knowledge of uterine receptivity in guinea pigs.

Comparative co-expression analysis of RNA-Seq transcriptome revealing key genes, miRNA and transcription factor in distinct metabolic pathways in diabetic nerve, eye, and kidney disease.

Diabetes and its related complications are associated with long term damage and failure of various organ systems. The microvascular complications of diabetes considered in this study are diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy. The aim is to identify the weighted co-expressed and differentially expressed genes (DEGs), major pathways, and their miRNA, transcription factors (TFs) and drugs interacting in all the three conditions. The primary goal is to identify vital DEGs in all the three conditions. The overlapped five genes (AKT1, NFKB1, MAPK3, PDPK1, and TNF) from the DEGs and the co-expressed genes were defined as key genes, which differentially expressed in all the three cases. Then the protein-protein interaction network and gene set linkage analysis (GSLA) of key genes was performed. GSLA, gene ontology, and pathway enrichment analysis of the key genes elucidates nine major pathways in diabetes. Subsequently, we constructed the miRNA-gene and transcription factor-gene regulatory network of the five gene of interest in the nine major pathways were studied. hsa-mir-34a-5p, a major miRNA that interacted with all the five genes. RELA, FOXO3, PDX1, and SREBF1 were the TFs interacting with the major five gene of interest. Finally, drug-gene interaction network elucidates five potential drugs to treat the genes of interest. This research reveals biomarker genes, miRNA, TFs, and therapeutic drugs in the key signaling pathways, which may help us, understand the processes of all three secondary microvascular problems and aid in disease detection and management.

The protective effects of curcumin on depression: Genes, transcription factors, and microRNAs involved

BackgroundWe aim to identify the molecular mechanisms for curcumin's anti-depressant properties, including genes, transcription factors, and miRNAs. MethodsThe Comparative Toxicogenomics Database, GeneMania, Metascape, MIENTURNET, and Cytoscape software were used as important data approaches in this study. ResultsCurcumin may have an anti-depressant effect via the relevant genes: ADORA2A, ALB, BDNF, FGF2, GLO1, GSK3B, IL6, MIF, NOS1, PTGS2, RELN, SELP, SOD1, and NR3C1. Co-expression (50.7 %) and physical interactions (28.7 %) were the primary relationships discovered by gene network analysis. The key pathways involved in curcumin's protective function against depression were “spinal cord injury”, “regulation of apoptotic signaling pathway”, “positive regulation of protein phosphorylation”, “folate metabolism”, “neuroinflammation and glutamatergic signaling”, and “inflammation response”. We also observed 74 miRNAs associated with depression that are targeted by curcumin, with hsa-miR-146a-5p having the greatest expression and interaction. PLSCR1, SNAI1, ZNF267, ATF3, and GTF2B were the most important transcription factors that regulated four curcumin-targeted genes. Curcumin's physicochemical characteristics and pharmacokinetics are consistent with its antidepressant effects due to its high gastrointestinal absorption, which did not remove it from the CNS, and its ability to penetrate the blood-brain barrier. Curcumin also inhibits CYP1A9 and CYP3A4. LimitationsA toxicogenomic design in silico was applied. ConclusionsOur findings suggest that therapy optimization and further research into curcumin's pharmacological properties are required before it may be utilized to treat depression.

PSXV-2 Genome-Wide Association and Gene Network Analysis of Fatty Acid Composition in Beef from Brangus Cattle

Abstract Beef is high in key nutrients; however, it is often perceived as unhealthy because of its high fat content, particularly its saturated fat. The fat content of beef is controlled by multiple genetic factors and impacts meat quality traits. The objectives of this study were to 1) characterize the natural variation in fatty acid (FA) composition of Bos taurus indicus influenced beef cattle, 2) estimate phenotypic and genetic correlations of FA composition with carcass and meat quality traits, 3) use genome-wide association (GWAS) to identify genes with the greatest effect on FA composition, and 4) use gene network analysis to identify genes with small effects on FA content. The following traits were recorded on 1,066 Brangus steer: carcass weight (kg; 373 ± 36.3); marbling (436 ± 84.2); quality grade; back fat (cm; 1.6 ± 0.6); ribeye area (cm2; 83.3 ± 9.5); yield grade (3.2 ± 0.9); Warner-Bratzler shear force (kg; 5.1 ± 1.0); and 27 FA [saturated (%; 47.6 ± 3.1), monounsaturated (%; 46.3 ± 3.5), and polyunsaturated (%; 6.1 ± 2.0)]. Carcass traits had weak beneficial correlations to FA composition suggesting the possibility to manage or select for beef with less saturated fat without negatively impacting meat quality. GWAS identified four QTL and gene network analysis identified 177 genes related to lipid storage, transportation, and modification.

Comprehensive analysis of fifteen hub genes to identify a promising diagnostic model, regulated networks, and immune cell infiltration in acute kidney injury.

Acute kidney injury is a common clinical problem with no sensitive and specific diagnostic biomarkers and definitive treatments. The underlying molecular mechanisms of acute kidney injury are unclear. Therefore, it is pivotal to explore the underlying mechanisms and screen for novel diagnostic biomarkers, and therapeutic targets. The present study identified 15 hub genes by WGCNA analysis. LASSO-based logistic regression analysis was used to select key features and construct a diagnostic model of AKI. In addition, GO and KEGG analyses were performed and TF-mRNA and miRNA-mRNA network analysis and immune infiltration analysis of hub genes were performed to reveal the underlying mechanisms of AKI. A diagnostic model was constructed by LASSO-based logistic regression analysis and was validated by RT-qPCR based on 15 hub genes. GO and KEGG analyses revealed DEGs were enriched in oxidation-reduction process, cell adhesion, proliferation, migration, and metabolic process. The enriched TFs were BRD2, EP300, ETS1, MYC, SPI1, and ZNF263. The enriched miRNAs were miR-181c-5p, miR-218-5p, miR-485-5p, miR-532-5p and miR-6884-5p. The immune infiltration analysis showed that Macrophages M2 was decreasing significantly revealing a protective factor for further AKI treatment. The present study identified 15 hub genes based on WGCNA. Development and validation of a potentially diagnostic model based on 15 hub genes. In addition, exploring the interaction between transcriptional factors and 15 hub genes, and miRNA-mRNA relationship pairs. Furthermore, immune infiltration analysis was performed by analyzing gene expression profiles of AKI. Our study provides some basis for further experimental studies.

Functional analysis of litter size and number of teats in pigs: From GWAS to post-GWAS

Reproductive traits, such as the number of teats and litter size, are essential for animal breeding programs due to the importance of the production chain, since they influence the maternal ability of the sow and can affect the number of weaned piglets. We aim to identify candidate genes associated with reproductive traits in pigs, using GWAS data from a systematic review combined with sequencing data, to build networks of biological processes and transcription factors (TFs) from the identified genes to highlight the most candidate genes for litter size and the number of teats. In the systematic review, only peer-reviewed articles were used, with descriptors related to the evaluated traits, and selected based on eligibility criteria. Fourteen papers were selected and classified for functional analysis of gene networks with 2077 candidate genes identified. After combining with the list of genes presenting known structural variants in the 5′UTR and/or coding region, 306 genes remained to be used to build the gene networks of biological processes and TFs, highlighting processes associated with litter size (e.g., ionotropic glutamate receptor signaling pathway and blastocyte growth) and the number of teats (e.g., growth hormone receptor, regulation of the BMP - Bone Morphogenetic Proteins signaling pathway and blood vessel proliferation). Two most candidate genes for litter size trait (GRID2 and PALB2) and six most candidate genes for the number of teats (GHR, IFT80, FSTL3, SKOR1, SMURF1, and AKT3) were prioritized. TFs associated with candidate genes were also identified for litter size (PALB2 and GRID2) and the number of teats (RIN, LTBP2, and COL6A6). Thus, it is suggested that the most candidate genes and TFs presented in this study may play an important role in the traits studied, being important for genetic studies and animal breeding.

Unraveling the Transcriptomic Signatures of Homologous Recombination Deficiency in Ovarian Cancers.

Homologous recombination deficiency (HRD) is a crucial driver of tumorigenesis by inducing impaired repair of double-stranded DNA breaks. Although HRD possibly triggers the production of numerous tumor neoantigens that sufficiently stimulate and activate various tumor-immune responses, a comprehensive understanding of the HRD-associated tumor microenvironment is elusive. To investigate the effect of HRD on the selective enrichment of transcriptomic signatures, 294 cases from The Cancer Genome Atlas-Ovarian Cancer project with both RNA-sequencing and SNP array data are analyzed. Differentially expressed gene analysis and network analysis are performed to identify HRD-specific signatures. Gene-sets associated with mitochondrial activation, including enhanced oxidative phosphorylation (OxPhos), are significantly enriched in the HRD-high group. Furthermore, a wide range of immune cell activation signatures is enriched in HRD-high cases of high-grade serous ovarian cancer (HGSOC). On further cell-type-specific analysis, M1-like macrophage genes are significantly enriched in HRD-high HGSOC cases, whereas M2-macrophage-related genes are not. The immune-response-associated genomic features, including tumor mutation rate, neoantigens, and tumor mutation burdens, correlated with HRD scores. In conclusion, the results of this study highlight the biological properties of HRD, including enhanced energy metabolism, increased tumor neoantigens and tumor mutation burdens, and consequent exacerbation of immune responses, particularly the enrichment of M1-like macrophages in HGSOC cases.

Co-overexpression of AtSAT1 and EcPAPR improves seed nutritional value in maize.

Maize seeds synthesize insufficient levels of the essential amino acid methionine (Met) to support animal and livestock growth. Serine acetyltransferase1 (SAT1) and 3′-phosphoadenosine-5′-phosphosulfate reductase (PAPR) are key control points for sulfur assimilation into Cys and Met biosynthesis. Two high-MET maize lines pRbcS:AtSAT1 and pRbcS:EcPAPR were obtained through metabolic engineering recently, and their total Met was increased by 1.4- and 1.57-fold, respectively, compared to the wild type. The highest Met maize line, pRbcS:AtSAT1-pRbcS:EcPAPR, was created by stacking the two transgenes, causing total Met to increase 2.24-fold. However, the pRbcS:AtSAT1-pRbcS:EcPAPR plants displayed progressively severe defects in plant growth, including early senescence, stunting, and dwarfing, indicating that excessive sulfur assimilation has an adverse effect on plant development. To explore the mechanism of correlation between Met biosynthesis in maize leaves and storage proteins in developing endosperm, the transcriptomes of the sixth leaf at stage V9 and 18 DAP endosperm of pRbcS:AtSAT1, pRbcS:AtSAT1-pRbcS:EcPAPR, and the null segregants were quantified and analyzed. In pRbcS:AtSAT1-pRbcS:EcPAPR, 3274 genes in leaves (1505 up- and 1769 downregulated) and 679 genes in the endosperm (327 up- and 352 downregulated) were differentially expressed. Gene ontology (GO) and KEGG (Kyoto encyclopedia of genes and genomes) analyses revealed that many genes were associated with Met homeostasis, including transcription factors and genes involved in cysteine and Met metabolism, glutathione metabolism, plant hormone signal transduction, and oxidation–reduction. The data from gene network analysis demonstrated that two genes, serine/threonine-protein kinase (CCR3) and heat shock 70 kDa protein (HSP), were localized in the core of the leaves and endosperm regulation networks, respectively. The results of this study provide insights into the diverse mechanisms that underlie the ideal establishment of enhanced Met levels in maize seeds.

Systems pharmacology dissection of the mechanisms and therapeutic potential of Cassiae semen for hepatoprotection and brightening eyes

Cassiae semen has been shown to play significant roles in reversing “liver fire” to improve vision. The systems mechanism of Cassiae semen for hepatoprotection and brightening eyes has not been fully explored. The systems pharmacology approach is proposed to dissect the potential pharmacological mechanism of Cassiae semen for hepatoprotection and brightening eyes. The results showed that 26 active components of Cassiae semen that connected with 230 targets were obtained. Gene ontology enrichment, network and pathway analysis explored that Cassiae semen is responsible for hepatoprotection and brightening eyes. The current study will contribute to the research and development of functional foods.

Expression profiling of mRNA and functional network analyses of genes regulated by human papilloma virus E6 and E7 proteins in HaCaT cells.

Human papillomavirus (HPV) oncogenes E6 and E7 are essential for HPV-related cancer development. Here, we developed a cell line model using lentiviruses for transfection of the HPV16 oncogenes E6 and E7 and investigated the differences in mRNA expression during cell adhesion and chemokine secretion. Subsequently, RNA sequencing (RNA-seq) analysis was performed to explore the differences in mRNA expression. Compared to levels in the control group, 2,905 differentially expressed mRNAs (1,261 downregulated and 1,644 upregulated) were identified in the HaCaT-HPV16E6E7 cell line. To predict the functions of these differentially expressed genes (DEGs) the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used. Protein–protein interactions were established, and the hub gene was identified based on this network. Real-time quantitative-PCR (RT-qPCR) was conducted to confirm the levels of 14 hub genes, which were consistent with the RNA-seq data. According to this, we found that these DEGs participate in the extracellular matrix (ECM), cell adhesion, immune control, and cancer-related signaling pathways. Currently, an increasing number of clinicians depend on E6/E7mRNA results to make a comprehensive judgment of cervical precancerous lesions. In this study, 14 hub genes closely related to the expression of cell adhesion ability and chemokines were analyzed in HPV16E6E7-stably expressing cell lines, which will open up new research ideas for targeting E6E7 in the treatment of HPV-related cancers.

Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain.

(1,3;1,4)-β-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located −331 bp to −382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.