Upstream Regulatory Network Research Articles

TTC7B triggers the PI4KA-AKT1-RXRA-FTO axis and inhibits colon cancer cell proliferation by increasing RNA methylation.

TTC7B is the PI4KA-binding protein. The upstream regulatory network associated with the expression of genes involved in RNA N6-adenine (m6A) methylation is not clear. Bioinformatics analysis revealed that the expression levels of TTC7B, PI4KA, and FTO are positively correlated with each other across human tissues. These genes are consistently downregulated in many cancers. We initially confirmed the correlation of the expression of these genes in colon cancer tissues from patients (n=105) and reported that TTC7B downregulation was significantly associated with poor prognosis. We subsequently performed a series of biological experiments and demonstrated that TTC7B upregulated RXRA expression probably through the PI4KA-mediated AKT1 pathway and that RXRA was a transcription factor for the FTO gene. TTC7B inhibited the proliferation of colon cancer cells by increasing the recruitment of RXRA to the FTO promoter, increasing FTO expression, and decreasing the total RNA m6A level. Ablation of FTO demethylase activity completely abolished the inhibitory effect of TTC7B on the proliferation of cancer cells in vitro and in vivo. In conclusion, our study demonstrated for the first time that TTC7B triggers the RXRA-FTO axis through PI4KA binding, which leads to a decrease in total RNA m6A modification and the inhibition of colon cancer progression.

Deciphering Immune-related Gene Signatures in Diabetic Retinopathy: Insights from In silico Analysis and In vitro Experiment.

Diabetes retinopathy (DR) is one of the most common microvascular consequences of diabetes, and the economic burden is increasing. Our aim is to decipher the relevant mechanisms of immune-related gene features in DR and explore biomarkers targeting DR. Provide a basis for the treatment and prevention of DR. The immune infiltration enrichment score of DR patients was evaluated from the single- cell RNA sequencing dataset, and the samples were divided into low immune subgroups and high immune subgroups based on this result. Through weighted gene correlation network analysis, differentially expressed genes (DEGs) between two subgroups were identified and crossed with genes with the strongest immune association, resulting in significant key genes. Then divide the DR individuals into two immune related differentially expressed gene (IDEG) clusters, A and B. Submit cross DEGs between two clusters through Gene Set Enrichment Analysis (GSEA) to further explore their functions. A protein-protein interaction (PPI) network of IDEG was established to further identify central genes associated with DR. Use the discovered central genes to predict the regulatory network involved in the pathogenesis of DR. Then, the role of the identified hub gene in the pathogenesis of DR was further studied through in vitro experiments. We found that the immune scores of DR and control groups were different, and 27 IDEGs were found in the DR subgroup. Compared with cluster A, the proportion of cytotoxic lymphocytes, B lineage, monocyte lineage, and fibroblasts in DR patients in cluster B is significantly enriched. GSEA indicates that these genes are associated with T cell activation, regulation of immune response processes, lymphocyte-mediated immunity, TNF signaling pathway, and other signaling pathways. The PPI network subsequently identified 10 hub genes in DR, including SIGLEC10, RGS10, PENK, FGD2, LILRA6, CIITA, EGR2, SIGLEC7, LILRB1, and CD300LB. The upstream regulatory network and lncRNA miRNA mRNA ceRNA network of these hub genes were ultimately constructed. The discovery and identification of these genes will provide biomarkers for targeted prediction and treatment of DR. By integrating bioinformatics analysis and in vitro experiments, we have identified a set of central genes, indicating that these genes can serve as potential biomarkers for DR, which may be promising targets for future DR immunotherapy interventions.

Transcriptomic analysis and validation study of key genes and the HIF‑1α/HO‑1 pathway associated with ferroptosis in neutrophilic asthma.

Ferroptosis, as a unique form of cell death caused by iron overload and lipid peroxidation, is involved in the pathogenesis of various inflammatory diseases of the airways. Inhibition of ferroptosis has become a novel strategy for reducing airway epithelial cell death and improving airway inflammation. The aim of the present study was to analyze and validate the key genes and signaling pathways associated with ferroptosis by bioinformatic methods combined with experimental analyzes in vitro and in vivo to aid the diagnosis and treatment of neutrophilic asthma. A total of 1,639 differentially expressed genes (DEGs) were identified in the transcriptome dataset. After overlapping with ferroptosis-related genes, 11 differentially expressed ferroptosis-related genes (DE-FRGs) were obtained. A new diagnostic model was constructed by these DE-FRGs from the transcriptome dataset with those from the GSE108417 dataset. The receiver operating characteristic curve analysis indicated that the area under the curve had good diagnostic performance (>0.8). As a result, four key DE-FRGs (CXCL2, HMOX1, IL-6 and SLC7A5) and biological pathway [hypoxia-inducible factor 1 (HIF-1) signaling pathway] associated with ferroptosis in neutrophilic asthma were identified by the bioinformatics analysis combined with experimental validation. The upstream regulatory network of key DE-FRGs and target drugs were predicted and the molecular docking results from screened 37 potential therapeutic drugs revealed that the 13 small-molecule drugs exhibited a higher stable binding to the primary proteins of key DE-FRGs. The results suggested that four key DE-FRGs and the HIF-1α/heme oxygenase 1 pathway associated with ferroptosis have potential as novel markers or targets for the diagnosis or treatment of neutrophilic asthma.

HDAC2 promotes autophagy-associated HCC malignant progression by transcriptionally activating LAPTM4B

Hepatocellular carcinoma (HCC) is a significant global health challenge. The activation of autophagy plays an essential role in promoting the proliferation and survival of cancer cells. However, the upstream regulatory network and mechanisms governing autophagy in HCC remain unclear. This study demonstrated that histone deacetylase 2 (HDAC2) regulates autophagy in HCC. Its expression was elevated in HCC tissues, and high HDAC2 expression was strongly associated with poor prognosis in individuals with HCC. Integrated in vitro and in vivo investigations confirmed that HDAC2 promotes autophagy and autophagy-related malignant progression in HCC. Mechanistically, HDAC2 bound specifically to the lysosome-associated protein transmembrane 4-β (LAPTM4B) promoter at four distinct binding sites, enhancing its transcriptional activation and driving autophagy-related malignant progression in HCC. These findings establish LAPTM4B as a direct target gene of HDAC2. Furthermore, the selective inhibitor of HDAC2 effectively alleviated the malignant development of HCC. In addition, multivariate Cox regression analysis of 105 human HCC samples revealed that HDAC2 expression is an independent predictor of HCC prognosis. This study underscores the crucial role of the HDAC2-LAPTM4B axis in regulating autophagy in the malignant evolution of HCC and highlights the potential of targeting HDAC2 to prevent and halt the malignant progression of HCC.

Jun/Fos promotes migration and invasion of hepatocellular carcinoma cells by enhancing BORIS promoter activity

The Brother of the Regulator of Imprinted Sites (BORIS), as a specific indicator of hepatocellular carcinoma, exhibits a significant increase in expression. However, its upstream regulatory network remains enigmatic. Previous research has indicated a strong correlation between the Hippo pathway and the progression of hepatocellular carcinoma. It is well established that the Activator Protein-1 (AP-1) frequently engages in interactions with the Hippo pathway. Thus, we attempt to prove whether Jun and Fos, a major member of the AP-1 family, are involved in the regulation of BORIS expression. Bioinformatics analysis revealed the existence of binding sites for Jun and Fos within the BORIS promoter. Through a series of overexpression and knockdown experiments, we corroborated that Jun and Fos have the capacity to augment BORIS expression, thereby fostering the migration and invasion of hepatocellular carcinoma cells. Moreover, Methylation-Specific PCR and Bisulfite Sequencing PCR assays revealed that Jun and Fos do not have a significant impact on the demethylation of the BORIS promoter. However, luciferase reporter and chromatin immunoprecipitation experiments substantiated that Jun and Fos could directly bind to the BORIS promoter, thereby enhancing its transcription. In conclusion, these results suggest that Jun and Fos can promote the development of hepatocellular carcinoma by directly regulating the expression of BORIS. These findings may provide experimental evidence positioning BORIS as a novel target for the clinical intervention of hepatocellular carcinoma.

Advances and Prospects in Understanding Vertebrate Cardiac Conduction System, Pacemaker Cell, and Cardiac Muscle Development: Toward Novel Biological Therapies

The heart is composed of muscle cells called cardiomyocytes, including a specialized population named pacemaker cells that form the cardiac conduction system (CCS), which is responsible for generating the action potential dictating heart contractions. Failure of the CCS system leads to cardiac arrhythmias, which require complicated therapies and often the surgical implantation of electrical pacemakers. However, recent research has focused on the development of novel therapies using biological pacemakers that aim to substitute electrical devices. While most signaling pathways and transcription factors involved in the development of the pacemaker cells are known, the upstream regulatory networks need to be predicted through computer-based databases, mathematical modeling, as well as the functional testing of the regulatory elements in vivo, indicating the need for further research. Here, we summarize the current knowledge about the vertebrate myocardial CCS system and the development of the pacemaker cells, as well as emphasize the areas of future research to clarify the regulation of muscle pacemaker cells and the ease of development of biological therapies.

Molecular identification of phenylalanine ammonia lyase-encoding genes EfPALs and EfPAL2-interacting transcription factors in Euryale ferox.

Flavonoids are one of the most important secondary metabolites in plants, and phenylalanine ammonia-lyase (PAL) is the first rate-limiting enzyme for their biosynthesis. However, detailed information on the regulation of PAL in plants is still little. In this study, PAL in E. ferox was identified and functionally analyzed, and its upstream regulatory network was investigated. Through genome-wide identification, we obtained 12 putative PAL genes from E. ferox. Phylogenetic tree and synteny analysis revealed that PAL in E. ferox was expanded and mostly preserved. Subsequently, enzyme activity assays demonstrated that EfPAL1 and EfPAL2 both catalyzed the production of cinnamic acid from phenylalanine only, with EfPAL2 exhibiting a superior enzyme activity. Overexpression of EfPAL1 and EfPAL2 in Arabidopsis thaliana, respectively, both enhanced the biosynthesis of flavonoids. Furthermore, two transcription factors, EfZAT11 and EfHY5, were identified by yeast one-hybrid library assays as binding to the promoter of EfPAL2, and further luciferase (LUC) activity analysis indicated that EfZAT11 promoted the expression of EfPAL2, while EfHY5 repressed the expression of EfPAL2. These results suggested that EfZAT11 and EfHY5 positively and negatively regulate flavonoid biosynthesis, respectively. Subcellular localization revealed that EfZAT11 and EfHY5 were localized in the nucleus. Our findings clarified the key EfPAL1 and EfPAL2 of flavonoid biosynthesis in E. ferox and established the upstream regulatory network of EfPAL2, which would provide novel information for the study of flavonoid biosynthesis mechanism.

Abstract P4-08-23: ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease in women, while the oncogenic mechanisms which contribute to TNBC development, progression, and metastasis are poorly investigated and therefore warrant the critical need to identify novel oncogenic signalings and develop new therapeutic targets. Results: We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α protein level and activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggesting that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles in controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC. Conclusion: ZHX2 has been highlighted as one critical hypoxia-related factors regulator contributing to triple-negative breast cancer in this work, which has enriched the upstream regulatory network of HIF-1α signaling. Moreover, identification of key residuals determining biological function of ZHX2 provides novel approaches for treating TNBC via targeting the hypoxia pathway. Citation Format: Chengheng Liao, Wentong Fang, Rachel Shi, Jeremy Simon, Travis Ptacek, Giada Zurlo, Youqiong Ye, Leng Han, Cheng Fan, Lei Bao, Christopher Llynard Ortiz, Hong-Rui Lin, Ujjawal Manocha, Weibo Luo, Yan Peng, William Y Kim, Lee-Wei Yang, Qing Zhang. ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-23.

The novel gene, mtre-1 , is expressed downstream of MAB-3 and DMD-3 in the male tail tip at the termination of male tail tip retraction.

The development of the adult C. elegans male tail involves an extensive remodeling during the last larval stage where the pointed tail of the L4 male is converted into the blunt-ended adult tail with its collection of mechano-sensitive rays. The first step in this remodeling is the retraction of the four hypodermal cells of the tail tip to generate the blunt-ended tail. Male tail tip retraction is an excellent model for characterizing how upstream regulatory networks interact with the downstream cell biological effectors that drive morphogenetic changes in all animals. Previously, we've shown that two DM-domain transcription factors, MAB-3 and DMD-3 , are central regulators of male tail tip retraction. Using a microarray-based approach we have identified ~400 genes that are more highly expressed in the L4 male tail tip relative to the hermaphrodite L4 tail tip. The uncharacterized gene T05H10.3 , which we've named mtre-1 , was highly over-represented in the male tail tip vs. the hermaphrodite tail tip and was under-represented in mab-3 ; dmd-3 mutant male tail tips vs. wild-type male tail tips. A transcriptional reporter for mtre-1 shows clear expression in the male tail tip cells for a short period (~3 hours) at the end of retraction. This expression is dependent on the activity of MAB-3 and DMD-3 , since expression is reduced in dmd-3 single mutant males and absent in mab-3 ; dmd-3 mutant males. Finally, males homozygous for a putative null allele of mtre-1 display a phenotypically wild-type adult male tail, indicating that mtre-1 is not essential for male tail morphogenesis.

Identification of PLAUR-related ceRNA and immune prognostic signature for kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) represents one of the most fatal cancers, usually showing malignant progression and a high tumor recurrence rate. The urokinase-type plasminogen activator receptor (PLAUR) plays a critical role in the initiation and progression of several cancers, including KIRC. However, the function and mechanism of PLAUR in patients with KIRC are still unclear and require further investigation. In the present study, we first explored the expression profile and prognostic values of PLAUR in pan-cancer based on The Cancer Genome Atlas and Genotype-Tissue Expression databases. PLAUR was upregulated in multiple cancers and was significantly associated with poor overall survival and disease-free survival only in patients with KIRC. Subsequently, the PVT1/SNHG15-hsa-miR-532-3p axis was identified as the most potential upstream regulatory network of PLAUR in KIRC. In addition, PLAUR expression was closely associated with tumor-infiltrating immune cells, tumor immunity biomarkers, and immunomodulator expression. Furthermore, we constructed a multiple-gene risk prediction signature according to the PLAUR-related immunomodulators (PRIs). A prognostic nomogram was then developed to predict the 1-, 3-, and 5-year survival probabilities of individuals. In conclusion, our study identified the PVT1/SNHG15-hsa-miR-532-3p-PLAUR axis and a prognostic signature of PRIs, which could be a reference for future clinical research.

Identification and Characterization of the Core Region of ZmDi19-5 Promoter Activity and Its Upstream Regulatory Proteins.

Drought-induced 19 (Di19) family genes play important roles in plant growth, development, and environmental stress responses. However, little is known about this family in maize. The upstream regulatory network of Di19 genes remains poorly understood in plant stress response, especially. In this study, seven ZmDi19 genes were identified, and sequence alignment, gene structure, and phylogenetic analysis was conducted. According to the phylogenetic analysis, the ZmDi19-5 promoter was cloned and multiple putative stress-responsive cis-acting elements (CAEs) were found in the promoter region. The transient transformation assay indicated that firefly luciferase (LUC)-expressed activity driven by the ZmDi19-5 promoter can be significantly induced by drought stress. A 450 bp core region of ZmDi19-5 promoter was identified, and 28 upstream regulatory proteins were screened using yeast one-hybird (Y1H) system. According to the functional annotation, some genes were related to photosynthesis, light response, and water transport, which may suggest the important roles of these genes in drought response. Particularly, five members that may be involved in drought response exhibited strong binding activity to the core region of the ZmDi19-5 promoter. This study laid an important foundation for further revealing the molecular mechanisms and regulatory network of Di19 genes in drought stress response.

Aberrant Gene Expression Profiling in Men With Sertoli Cell-Only Syndrome.

Sertoli cell-only syndrome (SCOS) is the most severe and common pathological type of non-obstructive azoospermia. The etiology of SCOS remains largely unknown to date despite a handful of studies reported in this area. According to the gene expression of testicular tissue samples in six datasets from the Gene Expression Omnibus, we detected 1441 differentially expressed genes (DEGs) between SCOS and obstructive azoospermia (OA) testicular tissue samples. Enriched GO terms and KEGG pathways for the downregulated genes included various terms and pathways related to cell cycle and reproduction, while the enrichment for the upregulated genes yielded many inflammation-related terms and pathways. In accordance with the protein-protein interaction (PPI) network, all genes in the most critical module belonged to the downregulated DEGs, and we obtained nine hub genes, including CCNB1, AURKA, CCNA2, BIRC5, TYMS, UBE2C, CDC20, TOP2A, and OIP5. Among these hub genes, six were also found in the most significant SCOS-specific module obtained from consensus module analysis. In addition, most of SCOS-specific modules did not have a consensus counterpart. Based on the downregulated genes, transcription factors (TFs) and kinases within the upstream regulatory network were predicted. Then, we compared the difference in infiltrating levels of immune cells between OA and SCOS samples and found a significantly higher degree of infiltration for most immune cells in SCOS than OA samples. Moreover, CD56bright natural killer cell was significantly associated with six hub genes. Enriched hallmark pathways in SCOS had remarkably more upregulated pathways than the downregulated ones. Collectively, we detected DEGs, significant modules, hub genes, upstream TFs and kinases, enriched downstream pathways, and infiltrated immune cells that might be specifically implicated in the pathogenesis of SCOS. These findings provide new insights into the pathogenesis of SCOS and fuel future advances in its theranostics.

Pathobiology and Therapeutic Relevance of GSK-3 in Chronic Hematological Malignancies.

Glycogen synthase kinase-3 (GSK-3) is an evolutionarily conserved, ubiquitously expressed, multifunctional serine/threonine protein kinase involved in the regulation of a variety of physiological processes. GSK-3 comprises two isoforms (α and β) which were originally discovered in 1980 as enzymes involved in glucose metabolism via inhibitory phosphorylation of glycogen synthase. Differently from other proteins kinases, GSK-3 isoforms are constitutively active in resting cells, and their modulation mainly involves inhibition through upstream regulatory networks. In the early 1990s, GSK-3 isoforms were implicated as key players in cancer cell pathobiology. Active GSK-3 facilitates the destruction of multiple oncogenic proteins which include β-catenin and Master regulator of cell cycle entry and proliferative metabolism (c-Myc). Therefore, GSK-3 was initially considered to be a tumor suppressor. Consistently, GSK-3 is often inactivated in cancer cells through dysregulated upstream signaling pathways. However, over the past 10–15 years, a growing number of studies highlighted that in some cancer settings GSK-3 isoforms inhibit tumor suppressing pathways and therefore act as tumor promoters. In this article, we will discuss the multiple and often enigmatic roles played by GSK-3 isoforms in some chronic hematological malignancies (chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, and B-cell non-Hodgkin’s lymphomas) which are among the most common blood cancer cell types. We will also summarize possible novel strategies targeting GSK-3 for innovative therapies of these disorders.

Hmx gene conservation identifies the origin of vertebrate cranial ganglia.

The evolutionary origin of vertebrates included innovations in sensory processing associated with the acquisition of a predatory lifestyle1. Vertebrates perceive external stimuli through sensory systems serviced by cranial sensory ganglia, whose neurons arise predominantly from cranial placodes; however, the understanding of the evolutionary origin of placodes and cranial sensory ganglia is hampered by the anatomical differences between living lineages and the difficulty in assigning homology between cell types and structures. Here we show that the homeobox transcription factor Hmx is a constitutive component of vertebrate sensory ganglion development and that in the tunicate Ciona intestinalis, Hmx is necessary and sufficient to drive the differentiation programme of bipolar tail neurons, cells previously thought to be homologues of neural crest2,3. Using Ciona and lamprey transgenesis, we demonstrate that a unique, tandemly duplicated enhancer pair regulated Hmx expression in the stem-vertebrate lineage. We also show notably robust vertebrate Hmx enhancer function in Ciona, demonstrating that deep conservation of the upstream regulatory network spans the evolutionary origin of vertebrates. These experiments demonstrate regulatory and functional conservation between Ciona and vertebrate Hmx, and point to bipolar tail neurons as homologues of cranial sensory ganglia.

Bioinformatics analysis reveals molecular connections between non-alcoholic fatty liver disease (NAFLD) and COVID-19.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has devastatingly impacted people’s lives. Non-alcoholic fatty liver disease (NAFLD) is fatal comorbidity of COVID-19 seen with potential risk factors to develop severe symptoms. This research focuses on determining and elucidating the molecular factors and connections that might contribute to the severity of SARS-CoV-2 infection in NAFLD patients. Here, we comprehensively inspected the genes involved in NAFLD and SARS-CoV-2 entry factors (SCEFs) found by searching through the DisGeNet database and literature review, respectively. Further, we identified the SCEFs-related proteins through protein-protein interaction (PPI) network construction, MCODE, and Cytohubba. Next, the shared genes involved in NAFLD and SARS-CoV-2 entry, and hub gene were determined, followed by the GO and KEGG pathways analysis. X2K database was used to construct the upstream regulatory network of hub genes, as well as to identify the top ten candidates of transcription factors (TFs) and protein kinases (PKs). PPI analysis identified connections between 4 top SCEFs, including ACE, ADAM17, DPP4, and TMPRSS2 and NAFLD-related genes such as ACE, DPP4, IL-10, TNF, and AKT1. GO and KEGG analysis revealed the top ten biological processes and pathways, including cytokine-mediated signaling, PI3K-Akt, AMPK, and mTOR signaling pathways. The upstream regulatory network revealed that AKT1 and MAPK14 as important PKs and HIF1A and SP1 as important TFs associated with AKT1, IL-10, and TNF. The molecular connections identified between COVID-19 and NAFLD may shed light on discovering the causes of the severity of SARS-CoV-2 infected NAFLD patients.

The Predictive Competing Endogenous RNA Regulatory Networks and Potential Prognostic and Immunological Roles of Cyclin A2 in Pan-Cancer Analysis.

Although accumulating evidence has verified the relationship between CCNA2 and cancers, no pan-cancer analysis about the function and the upstream molecular mechanism of CCNA2 is available. For the first time, we analyzed potential oncogenic roles of CCNA2 in 33 cancer types via The Cancer Genome Atlas (TCGA) database. Overexpression of CCNA2 is widespread in almost all cancer types, and it is related to poor prognosis and advanced pathological stages in most cases. Moreover, we conducted upstream miRNAs and lncRNAs of CCNA2 to establish upstream regulatory networks in kidney renal clear cell carcinoma (LINC00997/miR-27b-3p/CCNA2), liver hepatocellular carcinoma (SNHG16, GUSBP11, FGD5-AS1, LINC00630, CD27-AS1, LINC00997/miR-22-3p/CCNA2, miR-29b-3p/CCNA2, miR-29c-3p/CCNA2, and miR-204-5p/CCNA2), and lung adenocarcinoma (miRNA-218-5p/CCNA2 and miR-204-5p/CCNA2) by expression analysis, survival analysis, and correlation analysis. The CCNA2 expression is positively correlated with Th2 cell infiltration and negatively correlated with CD4+ central memory and effector memory T-cell infiltration in different cancer types. Furthermore, CCNA2 is positively associated with expressions of immune checkpoints (CD274, CTLA4, HAVCR2, LAG3, PDCD1, and TIGIT) in most cancer types. Our first CCNA2 pan-cancer study contributes to understanding the prognostic and immunological roles and potential upstream molecular mechanisms of CCNA2 in different cancers.

Abstract 13322: Development of the Preclinical Science Integration and Translation (PRESCIANT) Method and Application to the Apolipoprotein E Knockout Mouse Atherosclerosis Model

Introduction: Animal models of human diseases are used extensively to interrogate molecular mechanisms in a reductionist fashion. We tested whether aggregation and integration of preclinical data can identify new causative pathways that faithfully model human disease mechanisms. We have termed this novel technique, the Preclinical Science Integration and Translation (PRESCIANT) method. Methods and Results: Data were extracted from 716 manuscripts in the journal Arteriosclerosis, Thrombosis, and Vascular Biology from 1995-2019 using the apolipoprotein E knockout mouse (ApoE-KO) to study atherosclerosis. We identified 360 unique studies in which genes were experimentally perturbed in ApoE-KO mice to impact atherosclerotic plaque size and/or composition. Impacts of the interventions on plaque size, inflammation, and lipid content were analyzed using Ingenuity Pathway Analysis. The top upstream regulatory network (sc-58125, a COX2 inhibitor) linked 37.2% (134) of the genes implicated in atherosclerosis into a single network. Further, Ingenuity Pathway Analysis identified TREM1 signaling, LXR/RXR activation, and renin-angiotensin signaling as the top 3 pathways associated with changes in atherosclerosis parameters. Specifically, early atherogenesis genes were enriched with pathways associated with inflammatory cell migration and infiltration (including TREM1) whereas late atherosclerosis genes were associated with cell metabolism and survival (including LXR/RXR activation). These two pathways were interrogated in a clinical cohort of 88,660 patients by testing for association between genetically predicted expression of the human homologs of mouse pathway genes and a composite phenotype composed of 27 human atherosclerosis diagnoses. There was a significant enrichment (p<0.01 for both pathways) in the number of human homologs associated with atherosclerosis, including 12 (57.2%) genes in the TREM1 pathway and 15 (53.6%) in the LXR/RXR pathway. Conclusion: PRESCIANT can successfully leverage decades of animal investigations to translate results from large-volume singular preclinical studies to make novel causal inferences into human disease.

IRegNet: an integrative Regulatory Network analysis tool for Arabidopsis thaliana.

Gene expression is delicately controlled via multilayered genetic and/or epigenetic regulatory mechanisms. Rapid development of the high-throughput sequencing (HTS) technology and its derivative methods including chromatin immunoprecipitation sequencing (ChIP-seq) and DNA affinity purification sequencing (DAP-seq) have generated a large volume of data on DNA-protein interactions (DPIs) and histone modifications on a genome-wide scale. However, the ability to comprehensively retrieve empirically validated upstream regulatory networks of genes of interest (GOIs) and genomic regions of interest (ROIs) remains limited. Here, we present integrative Regulatory Network (iRegNet), a web application that analyzes the upstream regulatory network for user-queried GOIs or ROIs in the Arabidopsis (Arabidopsis thaliana) genome. iRegNet covers the largest empirically proven DNA-binding profiles of Arabidopsis transcription factors (TFs) and non-TF proteins, and histone modifications obtained from all currently available Arabidopsis ChIP-seq and DAP-seq data. iRegNet not only catalogs upstream regulomes and epigenetic chromatin states for single-query gene/genomic region but also suggests significantly overrepresented upstream genetic regulators and epigenetic chromatin states of user-submitted multiple query genes/genomic regions. Furthermore, gene-to-gene coexpression index and protein-protein interaction information were also integrated into iRegNet for a more reliable identification of upstream regulators and realistic regulatory networks. Thus, iRegNet will help discover upstream regulators as well as molecular regulatory networks of GOI(s) and/or ROI(s), and is freely available at http://chromatindynamics.snu.ac.kr:8082/iRegNet_main.

MicroRNA-128 inhibits mitochondrial biogenesis and function via targeting PGC1α and NDUFS4

The size and morphology of mitochondria are very heterogeneous and correlates well with their healthy functioning. In many pathological conditions, mitochondrial morphology is altered due to impaired mitochondrial dynamics (a collective term for mitochondrial fusion and fission) and dysfunction. The current study aimed at identifying the role of microRNA-128 (miR-128) in regulating mitochondrial biogenesis. Previously, peroxisome proliferator activator receptor γ coactivator 1α (PGC1α) has been shown to co-activate key intermediates of mitochondrial biogenesis, function, and dynamics; however, the upstream regulatory network remains largely unknown. We, herein using in silico analysis followed by in vitro experiments in C2C12 myoblasts, showed that miR-128 reduces mitochondrial biogenesis by directly targeting PGC1α. The expression of downstream genes, nuclear respiratory factors 1 and 2 (NRF1 and NRF2, respectively), and mitochondrial transcription factor A (TFAM) were decreased in C2C12 myoblasts upon overexpression of miR-128. Also, miR-128 is shown to promote mitochondrial dysfunction by directly targeting NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (NDUFS4). The mitochondrial dynamics and morphology were impaired post miR-128 overexpression, as revealed by downregulation of fusion proteins (mitofusin1 and 2, i.e., MFN1 and MFN2, respectively) and upregulation of fission protein (dynamin-related protein 1, i.e., DRP1). Conversely, inhibition of miR-128 expression improved mitochondrial biogenesis, function, and dynamics, as evidenced by increased mitochondrial mass and ATP production after antimiR-128 treatment. Our findings reveal that inhibition of miR-128 can be a new potential target for reversing the effects of metabolic disorders of skeletal muscle as observed during many pathophysiological conditions such as obesity and type II diabetes.

Single-cell profiling of tumor-infiltrating TCF1/TCF7+ T cells reveals a T lymphocyte subset associated with tertiary lymphoid structures/organs and a superior prognosis in oral cancer

ObjectivesDespite substantial advances in treatment, clinical outcomes for oral squamous cell carcinoma (OSCC) remain unsatisfactory. Tumor-infiltrating lymphocytes (TILs) are an important prognostic factor for patients and are heterogeneous. Some studies have suggested that TCF1/TCF7+ T cells and tertiary lymphatic structure/organ (TLS) play an important role in tumor immunity. However, how they affect tumor immunity and whether they are related to prognosis in OSCC have not been reported in detail. Materials and methodsWe isolated OSCC cells and performed single-cell RNA sequencing (scRNA-seq). We used immunohistochemistry (IHC) to analyze the relationship between TLSs and prognosis. Multiplex immunohistochemistry (MIHC), flow cytometry (FCM) and spatial analysis were performed to verify the characteristics of TCF1/TCF7+ T cells. The prognostic significance and upstream regulatory network of the TCF1/TCF7+ T cell subpopulation were determined by multivariate analysis and Scenic software. ResultsWe found a strong association between TCF1/TCF7+ T cell subsets, TLSs and prognosis. The results suggested that TCF1/TCF7+ T cells express high levels of TLS-related genes and low levels of immune checkpoint molecules. Finally, we found that TCF1/TCF7+ T cells were significantly associated with favorable outcomes. We also describe the upstream drivers that these cells rely on. ConclusionsTCF1/TCF7+ T cells could be used as a new therapeutic target to regulate the immune response of OSCC and are expected to be a new prognostic marker.