Dihydroartemisinin Attenuates Hypoxia-Induced Pulmonary Hypertension Through the ELAVL2/miR-503/PI3K/AKT Axis.

ABSTRACT: Dihydroartemisinin (DHA) is an active form of artemisinin extracted from the traditional Chinese medicine Artemisia annua , which is used to treat malaria. Previous studies have shown that DHA has a therapeutic effect on pulmonary hypertension (PH), but its specific mechanism has not been fully elucidated. In this study, a hypoxia-induced PH mouse model was established and DHA was administered as a therapeutic intervention. We measured hemodynamics and right ventricular hypertrophy and observed hematoxylin and eosin staining of lung tissue sections, proving the therapeutic effect of DHA on PH. Furthermore, cell counting kit-8 and 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay kit were performed to examine cell proliferation of pulmonary artery smooth muscle cells cultured in hypoxia or in normoxia. Transwell migration chamber assay was performed to examine cell migration of the same cell model. Consistent with the therapeutic effect in vivo, DHA inhibited hypoxia-induced cell proliferation and migration. Through high-throughput sequencing of mouse lung tissue, we screened embryonic lethal abnormal vision-like 2 (ELAVL2) as a key RNA binding protein in PH. Mechanistically, DHA inhibited the proliferation and migration of pulmonary artery smooth muscle cells by promoting the expression of ELAVL2 and regulating the miR-503/PI3K/AKT pathway. The binding relationship between ELAVL2 and pre-miR-503 was verified by RNA binding protein immunoprecipitation assay. In conclusion, we first propose that DHA alleviates PH through the ELAVL2/miR-503/PI3K/AKT pathway, which may provide a basis for new therapeutic strategies of PH.

UBL5/Hub1: An Atypical Ubiquitin-Like Protein with a Typical Role as a Stress-Responsive Regulator.

Members of the ubiquitin-like protein family are known for their ability to modify substrates by covalent conjugation. The highly conserved ubiquitin relative UBL5/Hub1, however, is atypical because it lacks a carboxy-terminal di-glycine motif required for conjugation, and the whole E1-E2-E3 enzyme cascade is likely absent. Though the conjugation-mediated role of UBL5/Hub1 is controversial, it undoubtedly functions by interacting non-covalently with its partners. Several interactors of UBL5/Hub1 identified to date have suggested broad stress-responsive functions of the protein, for example, stress-induced control of pre-mRNA splicing, Fanconi anemia pathway of DNA damage repair, and mitochondrial unfolded protein response. While having an atypical mode of function, UBL5/Hub1 is still a stress protein that regulates feedback to various stimuli in a similar manner to other ubiquitin-like proteins. In this review, I discuss recent progress in understanding the functions of UBL5/Hub1 and the fundamental questions which remain to be answered.

HuB and HuD repress telomerase activity by dissociating HuR from TERC.

The ubiquitous RNA-binding protein HuR (ELAVL1) promotes telomerase activity by associating with the telomerase noncoding RNA TERC. However, the role of the neural-specific members HuB, HuC, and HuD (ELAVL2-4) in telomerase activity is unknown. Here, we report that HuB and HuD, but not HuC, repress telomerase activity in human neuroblastoma cells. By associating with AU-rich sequences in TERC, HuB and HuD repressed the assembly of the TERT-TERC core complex. Furthermore, HuB and HuD competed with HuR for binding to TERC and antagonized the function of HuR that was previously shown to enhance telomerase activity to promote cell growth. Our findings reveal a novel mechanism controlling telomerase activity in human neuroblastoma cells that involves a competition between HuR and the related, neural-specific proteins HuB and HuD.

Global analysis of RNA-protein interactions in TNF-α induced alternative splicing in metabolic disorders.

In this report, using the database of RNA-binding protein specificities (RBPDB) and our previously published RNA-seq data, we analyzed the interactions between RNA and RNA-binding proteins to decipher the role of alternative splicing in metabolic disorders induced by TNF-α. We identified 13 395 unique RNA-RBP interactions, including 385 unique RNA motifs and 35 RBPs, some of which (including MBNL-1 and 3, ZFP36, ZRANB2, and SNRPA) are transcriptionally regulated by TNF-α. In addition to some previously reported RBPs, such as RBMX and HuR/ELAVL1, we found a few novel RBPs, such as ZRANB2 and SNRPA, to be involved in the regulation of metabolic syndrome-associated genes that contain an enrichment of tetrameric RNA sequences (AUUU). Taken together, this study paves the way for novel RNA-protein interaction-based therapeutics for treating metabolic syndromes.

Investigation of hub genes and immune status in heart transplant rejection using endomyocardial biopsies.

T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) are severe post-transplantation complications for heart transplantation (HTx), whose molecular and immunological pathogenesis remains unclear. In the present study, the mRNA microarray data set GSE124897 containing 645 stable, 52 TCMR and 144 ABMR endomyocardial biopsies was obtained to screen for differentially expressed genes (DEGs) between rejected and stable HTx samples and to investigate immune cell infiltration. Functional enrichment analyses indicated roles of the DEGs primarily in immune-related mechanisms. Protein-protein interaction networks were then constructed, and ICAM1, CD44, HLA-A and HLA-B were identified as hub genes using the maximal clique centrality method. Immune cell infiltration analysis revealed differences in adaptive and innate immune cell populations between TCMR, ABMR and stable HTx samples. Additionally, hub gene expression levels significantly correlated with the degree and composition of immune cell infiltration in HTx rejection samples. Furthermore, drug-gene interactions were constructed, and 12 FDA-approved drugs were predicted to target hub genes. Finally, an external GSE2596 data set was used to validate the expression of the hub genes, and ROC curves indicated all four hub genes had promising diagnostic value for HTx rejection. This study provides a comprehensive perspective of molecular and immunological regulatory mechanisms underlying HTx rejection.

Genome-wide study of key genes and scoring system as potential noninvasive biomarkers for detection of suicide behavior in major depression disorder.

Although some progress has been made in the molecular biological detection of major depression disorder (MDD), its specificity and accuracy are still insufficient. This study is aimed to find hub genes, which could contribute to MDD related suicide and provide potential therapeutic targets for diagnosis and treatment. We downloaded RNA expression and clinical information from Gene Expression Omnibus (GEO) Dataset. Then, weighted gene co-expression network analysis (WGCNA) was applied to find core modules. Logistic regression was performed to identify the independent risk factors, and a scoring system was constructed based on these independent risk factors. As a result, a total of 16487 genes were selected to further conducted WGCNA analysis. We found that tan and green functional modules were exhibited high correlation with suicide behavior. 309 genes were identified in tan modules that were the strongest positively correlated with suicide behavior. Functional analysis in tan module indicated that activation of enzymes including nitric-oxide synthase and endoribonuclease, estrogen signaling pathway, glucagon signaling pathway, and legionellosis pathway were most enriched in MDD. Furthermore, we applied protein-protein interaction (PPI) analysis to select the hub genes and 10 genes were found in the core area of network. Then, we identified three-gene base independent risk signature by logistic regression model, including HSPA1A, RASEF, TBC1D8B. In conclusion, our study suggests that the tan module genes are closely related to suicide behaviors, which is mainly caused by multiple signaling pathway activation. The three-genes-based signature could provide a better efficacy to predict suicidal behavior in MDD patients.

Screening of hub genes and prediction of putative drugs in arsenic-related bladder carcinoma: An in silico study.

BACKGROUND: Evidence showed that inorganic arsenic (iAs) can trigger malignant transformation in cells with complex mechanisms. Thus, we aimed to investigate the possible molecules, pathways and therapeutic drugs for iAs-induced bladder cancer (BC) by using bioinformatics approaches. METHODS: Microarray-based data were analyzed to screen the differentially expressed genes (DEGs) between iAs-related BC cells and controls. Then, the roles of DEGs were annotated and the hub genes were screened out by protein-protein interaction network. The key genes were further selected from the hub genes through an assessment of the prognostic values. Afterward, potential drugs were predicted by using CMAP analysis. RESULTS: Analysis of a dataset (GSE90023) generated 21 upregulated and 47 downregulated DEGs, which were enriched in various signaling pathways. Among the DEGs, four hub genes including WNT7B, SFRP1, DNAJB2, and ATF3, were identified as the key genes because they might predict poor prognosis in BC patients. Lastly, Cantharidin was predicted to be a potential drug reversing iAs-induced malignant transformation in urinary epithelium cells. CONCLUSION: The present study found several hub genes involved in iAs-induced malignant transformation in urinary epithelium cells, and predicted several small agents for iAs toxicity prevention or therapy.

Targeting hub genes and pathways of innate immune response in COVID-19: A network biology perspective.

The current pandemic of 2019 novel coronavirus disease (COVID-19) caused by a novel virus strain, 2019-nCoV/SARS-CoV-2 have posed a serious threat to global public health and economy. It is largely unknown how the human immune system responds to this infection. A better understanding of the immune response to SARS-CoV-2 will be important to develop therapeutics against COVID-19. Here, we have used transcriptomic profile of human alveolar adenocarcinoma cells (A549) infected with SARS-CoV-2 and employed a network biology approach to generate human-virus interactome. Network topological analysis discovers 15 SARS-CoV-2 targets, which belongs to a subset of interferon (IFN) stimulated genes (ISGs). These ISGs (IFIT1, IFITM1, IRF7, ISG15, MX1, and OAS2) can be considered as potential candidates for drug targets in the treatments of COVID-19. We have identified significant interaction between ISGs and TLR3 agonists, like poly I: C, and imiquimod, and suggests that TLR3 agonists can be considered as a potential drug for drug repurposing in COVID-19. Our network centric analysis suggests that moderating the innate immune response is a valuable approach to target COVID-19.

ISG20 serves as a potential biomarker and drives tumor progression in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies and lacks reliable biomarkers for diagnosis and prognosis, which results in high incidence and mortality rates of ccRCC. In this study, ISG20, HJURP, and FOXM1 were identified as hub genes via weighted gene co-expression network analysis (WGCNA) and Cox regression analysis. Samples validation showed that only ISG20 was up-regulated in ccRCC. Therefore, ISG20 was selected for further study. High ISG20 expression was associated with poor overall survival and disease-free survival. Furthermore, the expression of ISG20 could effectively differentiate ccRCC from normal tissues and was positively correlated to clinical stages. Functional experiments proved that knockdown of ISG20 expression could obviously inhibit cell growth, migration, and invasion in ccRCC cells. To find the potential mechanisms of ISG20, gene set enrichment analysis (GSEA) was performed and revealed that high expression of ISG20 was significantly involved in metastasis and cell cycle pathways. In addition, we found that ISG20 could regulate the expression of MMP9 and CCND1. In conclusion, these findings suggested that ISG20 promoted cell proliferation and metastasis via regulating MMP9/CCND1 expression and might serve as a potential biomarker and therapeutic target in ccRCC.

ELAVL2-directed RNA regulatory network drives the formation of quiescent primordial follicles.

Formation of primordial follicles is a fundamental, early process in mammalian oogenesis. However, little is known about the underlying mechanisms. We herein report that the RNA-binding proteins ELAVL2 and DDX6 are indispensable for the formation of quiescent primordial follicles in mouse ovaries. We show that Elavl2 knockout females are infertile due to defective primordial follicle formation. ELAVL2 associates with mRNAs encoding components of P-bodies (cytoplasmic RNP granules involved in the decay and storage of RNA) and directs the assembly of P-body-like granules by promoting the translation of DDX6 in oocytes prior to the formation of primordial follicles. Deletion of Ddx6 disturbs the assembly of P-body-like granules and severely impairs the formation of primordial follicles, indicating the potential importance of P-body-like granules in the formation of primordial follicles. Furthermore, Ddx6-deficient oocytes are abnormally enlarged due to misregulated PI3K-AKT signaling. Our data reveal that an ELAVL2-directed post-transcriptional network is essential for the formation of quiescent primordial follicles.

RNA-Binding Proteins HuB, HuC, and HuD are Distinctly Regulated in Dorsal Root Ganglia Neurons from STZ-Sensitive Compared to STZ-Resistant Diabetic Mice.

The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.

Antidepressant-like actions by silencing of neuronal ELAV-like RNA-binding proteins HuB and HuC in a model of depression in male mice.

Currently available antidepressant drugs often fail to achieve full remission and patients might evolve to treatment resistance, showing the need to achieve a better therapy of depressive disorders. Increasing evidence supports that post-transcriptional regulation of gene expression is important in neuronal development and survival and a relevant role is played by RNA binding proteins (RBP). To explore new therapeutic strategies, we investigated the role of the neuron-specific ELAV-like RBP (HuB, HuC, HuD) in a mouse model of depression. In this study, a 4-week unpredictable chronic mild stress (UCMS) protocol was applied to mice to induce a depressive-like phenotype. In the last 2 weeks of the UCMS regimen, silencing of HuB, HuC or HuD was performed by using specific antisense oligonucleotides (aODN). Treatment of UCMS-exposed mice with anti-HuB and anti-HuC aODN improved both anhedonia and behavioural despair, used as measures of depressive-like behaviour, without modifying the response of stressed mice to an anxiety-inducing environment. On the contrary, HuD silencing promoted an anxiolytic-like behaviour in UCMS-exposed mice without improving depressive-like behaviours. The antidepressant-like phenotype of anti-HuB and anti-HuC mice was not shown concurrently with the promotion of adult hippocampal neurogenesis in the dentate gyrus, and no increase in the BDNF and CREB content was detected. Conversely, in the CA3 hippocampal region, projection area of newly born neurons, HuB and HuC silencing increased the number of BrdU/NeuN positive cells. These results give the first indication of a role of nELAV in the modulation of emotional states in a mouse model of depression.

Gene expression profiles and protein-protein interaction networks in amyotrophic lateral sclerosis patients with C9orf72 mutation.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that involves the death of neurons. ALS is associated with many gene mutations as previously studied. In order to explore the molecular mechanisms underlying ALS with C9orf72 mutation, gene expression profiles of ALS fibroblasts and control fibroblasts were subjected to bioinformatics analysis. Genes with critical functional roles can be detected by a measure of node centrality in biological networks. In gene co-expression networks, highly connected genes called as candidate hubs have been associated with key disease-related pathways. Herein, this method was applied to find the hub genes related to ALS disease. METHODS: Illumina HiSeq microarray gene expression dataset GSE51684 was retrieved from Gene Expression Omnibus (GEO) database which included four Sporadic ALS, twelve Familial ALS and eight control samples. Differentially Expressed Genes (DEGs) were identified using the Student's t test statistical method and gene co-expression networking. Gene ontology (GO) function and KEGG pathway enrichment analysis of DEGs were performed using the DAVID online tool. Protein-protein interaction (PPI) networks were constructed by mapping the DEGs onto protein-protein interaction data from publicly available databases to identify the pathways where DEGs are involved in. PPI interaction network was divided into subnetworks using MCODE algorithm and was analyzed using Cytoscape. RESULTS: The results revealed that the expression of DEGs was mainly involved in cell adhesion, cell-cell signaling, Extra cellular matrix region GO processes and focal adhesion, neuroactive ligand receptor interaction, Extracellular matrix receptor interaction. Tumor necrosis factor (TNF), Endothelin 1 (EDN1), Angiotensin (AGT) and many cell adhesion molecules (CAM) were detected as hub genes that can be targeted as novel therapeutic targets for ALS disease. CONCLUSION: These analyses and findings enhance the understanding of ALS pathogenesis and provide references for ALS therapy.

Mining topological structures of protein-protein interaction networks for human brain-specific genes.

Compared to other placental mammals, humans have unique thinking and cognitive abilities because of their developed cerebral cortex composed of billions of neurons and synaptic connections. As the primary effectors of the mechanisms of life, proteins and their interactions form the basis of cellular and molecular functions in the living body. In this paper, we developed a pipeline for mining topological structures, identifying functional modules, and analyzing their functions from publically available datasets. A human brain-specific protein-protein interaction network with 1482 nodes and 3105 edges was built using a MapReduce based shortest path algorithm. Within this, 7 functional cliques were identified using a network clustering method, 98 hub proteins were obtained by the calculation of betweenness and connectivity, and 5 closest relationship to clique connector proteins were recognized by the combination scores of topological distance and gene ontology similarity. Furthermore, we discovered functional modules interacting with TP53 protein, which involves several fragmented research study conclusions and might be an important clue for further in vivo or in silico experiments to confirm these associations.

Acute reduction of neuronal RNA binding Elavl2 protein and Gap43 mRNA in mouse hippocampus after kainic acid treatment.

Activity-dependent gene regulation in neurons has been hypothesized to be under transcriptional control and to include dramatic increases in immediate early genes (IEGs) after neuronal activity. In addition, several reports have focused on post-transcriptional regulation, which could be mediated by neuronal post-transcriptional regulators, including RNA binding proteins (RNABPs). One such protein family is the neuronal Elavls (nElavls; Elavl2, Elavl3, and Elavl4), whose members are widely expressed in peripheral and central nervous system. Previous reports showed that Elavl3 and 4 are up-regulated following repeated stimulation such as during cocaine administration, a seizure, or a spatial discrimination task. In this study, we focused on Elavl2, a candidate gene for schizophrenia and studied its role in neuronal activity. First we found that Elavl2 has a cell-type specific expression pattern that is highly expressed in hippocampal CA3 pyramidal neurons and hilar interneurons using Elavl2 specific antibody. Second, unexpectedly, we discovered that the Elavl2 protein level in the hippocampus was acutely down-regulated for 3 h after a kainic acid (KA)-induced seizure in the hippocampal CA3 region. In addition, level of Gap43 mRNA, a target mRNA of Elavl2 is decreased 12 h after KA treatment, thus suggesting the involvement of Elavl2 in activity-dependent RNA regulation.

An oocyte-specific ELAVL2 isoform is a translational repressor ablated from meiotically competent antral oocytes.

At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2°), which acts as a translational repressor. ELAVL2° is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2° overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2° levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence.