Hypoxia in the Blue Mussel Mytilus chilensis Induces a Transcriptome Shift Associated with Endoplasmic Reticulum Stress, Metabolism, and Immune Response.

The increase in hypoxia events, a result of climate change in coastal and fjord ecosystems, impacts the health and survival of mussels. These organisms deploy physiological and molecular responses as an adaptive mechanism to maintain cellular homeostasis under environmental stress. However, the specific effects of hypoxia on mussels of socioeconomic interest, such as Mytilus chilensis, are unknown. Using RNA-seq, we investigated the transcriptomic profiles of the gills, digestive gland, and adductor muscle of M. chilensis under hypoxia (10 days at 2 mg L-1) and reoxygenation (10 days at 6 mg L-1). There were 15,056 differentially expressed transcripts identified in gills, 11,864 in the digestive gland, and 9862 in the adductor muscle. The response varied among tissues, showing chromosomal changes in Chr1, Chr9, and Chr10 during hypoxia. Hypoxia regulated signaling genes in the Toll-like, mTOR, citrate cycle, and apoptosis pathways in gills, indicating metabolic and immunological alterations. These changes suggest that hypoxia induced a metabolic shift in mussels, reducing reliance on aerobic respiration and increasing reliance on anaerobic metabolism. Furthermore, hypoxia appeared to suppress the immune response, potentially increasing disease susceptibility, with negative implications for the mussel culture industry and natural bed populations. This study provides pivotal insights into metabolic and immunological adaptations to hypoxia in M. chilensis, offering candidate genes for adaptive traits.

The dynamic transcriptomic response of the goldfish brain under chronic hypoxia.

Oxygen is essential to fuel aerobic metabolism. Some species evolved mechanisms to tolerate periods of severe hypoxia and even anoxia in their environment. Among them, goldfish (Carassius auratus) are unique, in that they do not enter a comatose state under severely hypoxic conditions. There is thus significant interest in the field of comparative physiology to uncover the mechanistic basis underlying hypoxia tolerance in goldfish, with a particular focus on the brain. Taking advantage of the recently published and annotated goldfish genome, we profile the transcriptomic response of the goldfish brain under normoxic (21 kPa oxygen saturation) and, following gradual reduction, constant hypoxic conditions after 1 and 4 weeks (2.1 kPa oxygen saturation). In addition to analyzing differentially expressed protein-coding genes and enriched pathways, we also profile differentially expressed microRNAs (miRs). Using in silico approaches, we identify possible miR-mRNA relationships. Differentially expressed transcripts compared to normoxia were either common to both timepoints of hypoxia exposure (n = 174 mRNAs; n = 6 miRs), or exclusive to 1-week (n = 441 mRNAs; n = 23 miRs) or 4-week hypoxia exposure (n = 491 mRNAs; n = 34 miRs). Under chronic hypoxia, an increasing number of transcripts, including those of paralogous genes, was downregulated over time, suggesting a decrease in transcription. GO-terms related to the vascular system, oxidative stress, stress signalling, oxidoreductase activity, nucleotide- and intermediary metabolism, and mRNA posttranscriptional regulation were found to be enriched under chronic hypoxia. Known 'hypoxamiRs', such as miR-210-3p/5p, and miRs such as miR-29b-3p likely contribute to posttranscriptional regulation of these pathways under chronic hypoxia in the goldfish brain.

Hypoxic Effects on Matrix Metalloproteinases' Expression in the Tumor Microenvironment and Therapeutic Perspectives.

The tumor microenvironment (TME) is characterized by an acidic pH and low oxygen concentrations. Hypoxia induces neoplastic cell evasion of the immune surveillance, rapid DNA repair, metabolic reprogramming, and metastasis, mainly as a response to the hypoxic inducible factors (HIFs). Likewise, cancer cells increase matrix metalloproteinases' (MMPs) expression in response to TME conditions, allowing them to migrate from the primary tumor to different tissues. Since HIFs and MMPs are augmented in the hypoxic TME, it is easy to consider that HIFs participate directly in their expression regulation. However, not all MMPs have a hypoxia response element (HRE)-HIF binding site. Moreover, different transcription factors and signaling pathways activated in hypoxia conditions through HIFs or in a HIF-independent manner participate in MMPs' transcription. The present review focuses on MMPs' expression in normal and hypoxic conditions, considering HIFs and a HIF-independent transcription control. In addition, since the hypoxic TME causes resistance to anticancer conventional therapy, treatment approaches using MMPs as a target alone, or in combination with other therapies, are also discussed.

Identification and analysis of key hypoxia- and immune-related genes in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM), an autosomal dominant genetic disease, is the main cause of sudden death in adolescents and athletes globally. Hypoxia and immune factors have been revealed to be related to the pathology of HCM. There is growing evidence of a role for hypoxia and inflammation as triggers and enhancers in the pathology in HCM. However, the role of hypoxia- and immune-related genes in HCM have not been reported. METHODS: Firstly, we obtained four HCM-related datasets from the Gene Expression Omnibus (GEO) database for differential expression analysis. Immune cells significantly expressed in normal samples and HCM were then screened by a microenvironmental cell population counter (MCP-counter) algorithm. Next, hypoxia- and immune-related genes were screened by the LASSO + support vector machine recursive feature elimination (SVM-RFE) and weighted gene co-expression network analysis (WGCNA). Single-gene enrichment analysis and expression validation of key genes were then performed. Finally, we constructed a competing endogenous RNA (ceRNA) network of key genes. RESULTS: In this study, 35 differentially expressed hypoxia genes were found. By using LASSO + SVM-RFE analysis, 10 more targets with differentially expressed hypoxia genes were identified. The MCP-count algorithm yielded five differentially expressed immune cells, and after assessing them for WGCNA characteristics, 612 immune genes were discovered. When hypoxia and immune genes were combined for cross-tabulation analysis, three hypoxia- and immune-related genes (ATP2A2, DDAH1, and OMA1) were identified. CONCLUSION: Based on hypoxia characteristic genes, three key genes were identified. These were also significantly related to immune activation, which proves a theoretical basis and reference value for studying the relationship between HCM and hypoxia and immunity.

Responses and modulation of the white shrimp Litopenaeus vannamei glutathione peroxidases 2 and 4 during hypoxia, reoxygenation and GPx4 knock-down.

Glutathione peroxidases (GPxs) are important antioxidant enzymes that act at distinct levels of the antioxidant defense. In vertebrates, there are several glutathione peroxidase (GPx) isoforms with different cellular and tissue distribution, but little is known about their interrelationships. The shrimp Litopenaeus vannamei is the main crustacean cultivated worldwide. It is affected by environmental stressors, including hypoxia and reoxygenation that cause reactive oxygen species accumulation. Thus, the antioxidant response modulation is key for shrimp resilience. Recently, several GPx isoforms genes were identified in the L. vannamei genome sequence, but their functions are just beginning to be studied. As in vertebrates, shrimp GPx isoforms can present differences in their antioxidant responses. Also, there could be interrelationships among the isoforms that may influence their responses. We evaluated shrimp GPx2 and GPx4 expressions during hypoxia, reoxygenation, and GPx4 knock-down using RNAi for silencing, as well as the enzymatic activity of total GPx and GPx4. Also, glutathione content in hepatopancreas was evaluated. GPx2 and GPx4 presented similar expression patterns during hypoxia and reoxygenation. Their expressions decreased during hypoxia and were reestablished in reoxygenation at 6 h in non-silenced shrimp. GPx2 expression was down-regulated by GPx4 knock-down, suggesting that GPx4 affects GPx2 expression. Total GPx activity changed in hypoxia and reoxygenation at 6 h but not at 12 h, while GPx4 activity was not affected by any stressor. The GSH/GSSG ratio in hepatopancreas indicated that at early hours, the redox status remains well-modulated but at 12 h it is impaired by hypoxia and reoxygenation.

The pyruvate kinase of the whiteleg shrimp Litopenaeus vannamei: Gene structure and responses to short term hypoxia.

The shrimp Litopenaeus vannamei is the main farmed crustacean worldwide. This shrimp suffers environmental changes in oxygen availability that affect its energy metabolism. Pyruvate kinase (PK) catalyzes the last reaction of glycolysis and is key for the regulation of glycolysis and gluconeogenesis. There is ample knowledge about mammalian PK, but in crustaceans, the information is very scarce. In this study, we analyzed in silico the structures of the PK gene and protein. Also, the effects of hypoxia on gene expression, enzymatic activity, glucose, and lactate in hepatopancreas and muscle were analyzed. The PK gene is 15,103 bp and contains 11 exons and 10 introns, producing four mRNA variants by alternative splicing and named PK1, PK2, PK3 and PK4, that results in two proteins with longer C-terminus and two with a 12 bp insertion. The promoter contains putative binding sites for transcription factors (TF) that are typically involved in stress responses. The deduced amino acid sequences contain the classic domains, binding sites for allosteric effectors and potential reversible phosphorylation residues. Protein modeling indicates a homotetramer with highly conserved structure. The effect of hypoxia for 6 and 12 h showed tissue-specific patterns, with higher expression, enzyme activity and lactate in muscle, but higher glucose in hepatopancreas. Changes in response to hypoxia were detected at 12 h in expression with induction in muscle and reduction in hepatopancreas, while enzyme activity was maintained, and glucose and lactate decreased. These results show rapid changes in expression and metabolites, while enzyme activity was maintained to cope with short-term hypoxia.

"What We Know and What We Do Not Know about Evolutionary Genetic Adaptation to High Altitude Hypoxia in Andean Aymaras".

Three well-studied populations living at high altitudes are Tibetans, Andeans (Aymaras and Quechuas), and Ethiopians. Unlike Tibetans and Ethiopians who have similar hemoglobin (Hb) levels as individuals living at sea level, Aymara Hb levels increase when living at higher altitudes. Our previous whole genome study of Aymara people revealed several selected genes that are involved in cardiovascular functions, but their relationship with Hb levels was not elucidated. Here, we studied the frequencies of known evolutionary-selected variants in Tibetan and Aymara populations and their correlation with high Hb levels in Aymara. We genotyped 177 Aymaras at three different altitudes: 400 m (Santa Cruz), 4000 m (La Paz), and 5000 m (Chorolque), and correlated the results with the elevation of residence. Some of the Tibetan-selected variants also exist in Aymaras, but at a lower prevalence. Two of 10 Tibetan selected variants of EPAS1 were found (rs13005507 and rs142764723) and these variants did not correlate with Hb levels. Allele frequencies of 5 Aymara selected SNPs (heterozygous and homozygous) at 4000 m (rs11578671_BRINP3, rs34913965_NOS2, rs12448902_SH2B1, rs10744822_TBX5, and rs487105_PYGM) were higher compared to Europeans. The allelic frequencies of rs11578671_BRINP3, rs34913965_NOS2, and rs10744822_SH2B1 were significantly higher for Aymaras living at 5000 m than those at 400 m elevation. Variant rs11578671, close to the BRINP3 coding region, correlated with Hb levels in females. Variant rs34913965 (NOS2) correlated with leukocyte counts. Variants rs12448902 (SH2B1) and rs34913965 (NOS2) associated with higher platelet levels. The correlation of these SNPs with blood cell counts demonstrates that the selected genetic variants in Aymara influence hematopoiesis and cardiovascular effects.

Phox2b mutation mediated by Atoh1 expression impaired respiratory rhythm and ventilatory responses to hypoxia and hypercapnia.

Mutations in the transcription factor Phox2b cause congenital central hypoventilation syndrome (CCHS). The syndrome is characterized by hypoventilation and inability to regulate breathing to maintain adequate O2 and CO2 levels. The mechanism by which CCHS impact respiratory control is incompletely understood, and even less is known about the impact of the non-polyalanine repeat expansion mutations (NPARM) form. Our goal was to investigate the extent by which NPARM Phox2b mutation affect (a) respiratory rhythm; (b) ventilatory responses to hypercapnia (HCVR) and hypoxia (HVR); and (c) number of chemosensitive neurons in mice. We used a transgenic mouse line carrying a conditional Phox2bΔ8 mutation (same found in humans with NPARM CCHS). We crossed them with Atoh1cre mice to introduce mutation in regions involved with respiratory function and central chemoreflex control. Ventilation was measured by plethysmograph during neonatal and adult life. In room air, mutation in neonates and adult did not greatly impact basal ventilation. However, Phox2bΔ8, Atoh1cre increased breath irregularity in adults. The HVR and HCVR were impaired in neonates. The HVR, but not HCVR, was still partially compromised in adults. The mutation reduced the number of Phox2b+/TH--expressing neurons as well as the number of fos-activated cells within the ventral parafacial region (also named retrotrapezoid nucleus [RTN] region) induced by hypercapnia. Our data indicates that Phox2bΔ8 mutation in Atoh1-expressing cells impaired RTN neurons, as well as chemoreflex under hypoxia and hypercapnia specially early in life. This study provided new evidence for mechanisms related to NPARM form of CCHS neuropathology.

Evaluation of the promoter methylation status of hypoxia factor 3A and interleukin-6 genes and expression levels of mir-130b and mir-146b in childhood obesity.

OBJECTIVE: Obesity, which causes many serious diseases, is increasing exponentially in childhood across the world. Epigenetic changes, as well as genetics, play an important role in the process of adipogenesis. Therefore, we aimed to examine the expression levels of obesity-related MicroRNA-130b and MicroRNA-146b and the methylation status of hypoxia factor 3A and interleukin-6 genes associated with obesity in children. METHODS: This study was performed with 98 individuals (49 obese children and 49 controls) whose DNA was isolated from peripheral blood. Gene promoter methylations were analyzed by methylation-specific Polymerase chain reaction. In addition, expression levels of MicroRNAs were determined by quantitative real-time Polymerase chain reaction in 30 children (15 obese children and 15 controls). RESULTS: Methylation status of interleukin-6 gene was 93.9% in obese children (n=46/49) and 100% (n=49/49) in control group (p>0.05). There was no methylation for hypoxia factor 3A gene (p>0.05). As a result of the study, there was no statistically significant difference in terms of methylation status for hypoxia factor 3A and interleukin-6 genes in the obese group compared to the control group. However, we found that expression levels of MicroRNA-130b (p<0.01) and MicroRNA-146b (p<0.001) were higher in the obese group. CONCLUSIONS: Results support that MicroRNA-130b and MicroRNA-146b are potential biomarkers for the prevention and early diagnosis of obesity. This is the first study on childhood obesity in the Middle Black Sea region of Turkey. We believe that the results obtained by expanding the studies in our country and neighboring countries will be more decisive.

Effect of Hypoxia in the Transcriptomic Profile of Lung Fibroblasts from Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by exacerbated extracellular matrix deposition that disrupts oxygen exchange. Hypoxia and its transcription factors (HIF-1α and 2α) influence numerous circuits that could perpetuate fibrosis by increasing myofibroblasts differentiation and by promoting extracellular matrix accumulation. Therefore, this work aimed to elucidate the signature of hypoxia in the transcriptomic circuitry of IPF-derived fibroblasts. To determine this transcriptomic signature, a gene expression analysis with six lines of lung fibroblasts under normoxia or hypoxia was performed: three cell lines were derived from patients with IPF, and three were from healthy donors, a total of 36 replicates. We used the Clariom D platform, which allows us to evaluate a huge number of transcripts, to analyze the response to hypoxia in both controls and IPF. The control's response is greater by the number of genes and complexity. In the search for specific genes responsible for the IPF fibroblast phenotype, nineteen dysregulated genes were found in lung fibroblasts from IPF patients in hypoxia (nine upregulated and ten downregulated). In this sense, the signaling pathways revealed to be affected in the pulmonary fibroblasts of patients with IPF may represent an adaptation to chronic hypoxia.

mir152 hypomethylation as a mechanism for non-syndromic cleft lip and palate.

Non-syndromic cleft lip with or without cleft palate (NSCLP), the most common human craniofacial malformation, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological, and epigenetic findings. Epigenetic variations associated with NSCLP have been identified; however, functional investigation has been limited. Here, we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a region in mir152 that is frequently hypomethylated in NSCLP cohorts (21-26%), leading to mir152 overexpression. mir152 overexpression in human neural crest cells led to downregulation of spliceosomal, ribosomal, and adherens junction genes. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial cartilage impairment. Also, we suggest that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogous palatal alterations. We therefore propose that mir152 hypomethylation, potentially induced by hypoxia in early development, is a novel and frequent predisposing factor to NSCLP.

Exploring the Expression of Pro-Inflammatory and Hypoxia-Related MicroRNA-20a, MicroRNA-30e, and MicroRNA-93 in Periodontitis and Gingival Mesenchymal Stem Cells under Hypoxia.

Hypoxia associated with inflammation are common hallmarks observed in several diseases, and it plays a major role in the expression of non-coding RNAs, including microRNAs (miRNAs). In addition, the miRNA target genes for hypoxia-inducible factor-1α (HIF-1α) and nuclear factor of activated T cells-5 (NFAT5) modulate the adaptation to hypoxia. The objective of the present study was to explore hypoxia-related miRNA target genes for HIF-1α and NFAT5, as well as miRNA-20a, miRNA-30e, and miRNA-93 expression in periodontitis versus healthy gingival tissues and gingival mesenchymal stem cells (GMSCs) cultured under hypoxic conditions. Thus, a case-control study was conducted, including healthy and periodontitis subjects. Clinical data and gingival tissue biopsies were collected to analyze the expression of miRNA-20a, miRNA-30e, miRNA-93, HIF-1α, and NFAT5 by qRT-PCR. Subsequently, GMSCs were isolated and cultured under hypoxic conditions (1% O2) to explore the expression of the HIF-1α, NFAT5, and miRNAs. The results showed a significant upregulation of miRNA-20a (p = 0.028), miRNA-30e (p = 0.035), and miRNA-93 (p = 0.026) in periodontitis tissues compared to healthy gingival biopsies. NFAT5 mRNA was downregulated in periodontitis tissues (p = 0.037), but HIF-1α was not affected (p = 0.60). Interestingly, hypoxic GMSCs upregulated the expression of miRNA-20a and HIF-1α, but they downregulated miRNA-93e. In addition, NFAT5 mRNA expression was not affected in hypoxic GMSCs. In conclusion, in periodontitis patients, the expression of miRNA-20a, miRNA-30e, and miRNA-93 increased, but a decreased expression of NFAT5 mRNA was detected. In addition, GMSCs under hypoxic conditions upregulate the HIF-1α and increase miRNA-20a (p = 0.049) expression. This study explores the role of inflammatory and hypoxia-related miRNAs and their target genes in periodontitis and GMSCs. It is crucial to determine the potential therapeutic target of these miRNAs and hypoxia during the periodontal immune-inflammatory response, which should be analyzed in greater depth in future studies.

Uncovering Signals of Positive Selection in Peruvian Populations from Three Ecological Regions.

Peru hosts extremely diverse ecosystems which can be broadly classified into the following three major ecoregions: the Pacific desert coast, the Andean highlands, and the Amazon rainforest. Since its initial peopling approximately 12,000 years ago, the populations inhabiting such ecoregions might have differentially adapted to their contrasting environmental pressures. Previous studies have described several candidate genes underlying adaptation to hypobaric hypoxia among Andean highlanders. However, the adaptive genetic diversity of coastal and rainforest populations has been less studied. Here, we gathered genome-wide single-nucleotide polymorphism-array data from 286 Peruvians living across the three ecoregions and analyzed signals of recent positive selection through population differentiation and haplotype-based selection scans. Among highland populations, we identify candidate genes related to cardiovascular function (TLL1, DUSP27, TBX5, PLXNA4, SGCD), to the Hypoxia-Inducible Factor pathway (TGFA, APIP), to skin pigmentation (MITF), as well as to glucose (GLIS3) and glycogen metabolism (PPP1R3C, GANC). In contrast, most signatures of adaptation in coastal and rainforest populations comprise candidate genes related to the immune system (including SIGLEC8, TRIM21, CD44, and ICAM1 in the coast; CBLB and PRDM1 in the rainforest; and BRD2, HLA-DOA, HLA-DPA1 regions in both), possibly as a result of strong pathogen-driven selection. This study identifies candidate genes related to human adaptation to the diverse environments of South America.

Cyclic Hypoxia Induces Transcriptomic Changes in Mast Cells Leading to a Hyperresponsive Phenotype after FcεRI Cross-Linking.

Mast cells (MCs) play important roles in tumor development, executing pro- or antitumoral functions depending on tumor type and tumor microenvironment (TME) conditions. Cyclic hypoxia (cyH) is a common feature of TME since tumor blood vessels fail to provide a continuous supply of oxygen to the tumor mass. Here, we hypothesized that the localization of MCs in cyH regions within solid tumors could modify their transcriptional profile and activation parameters. Using confocal microscopy, we found an important number of MCs in cyH zones of murine melanoma B16-F1 tumors. Applying microarray analysis to examine the transcriptome of murine bone-marrow-derived MCs (BMMCs) exposed to interleaved cycles of hypoxia and re-oxygenation, we identified altered expression of 2512 genes. Functional enrichment analysis revealed that the transcriptional signature of MCs exposed to cyH is associated with oxidative phosphorylation and the FcεRI signaling pathway. Interestingly, FcεRI-dependent degranulation, calcium mobilization, and PLC-γ activity, as well as Tnf-α, Il-4, and Il-2 gene expression after IgE/antigen challenge were increased in BMMCs exposed to cyH compared with those maintained in normoxia. Taken together, our findings indicate that cyH causes an important phenotypic change in MCs that should be considered in the design of inflammation-targeted therapies to control tumor growth.

The close interaction between hypoxia-related proteins and metastasis in pancarcinomas.

Many primary-tumor subregions exhibit low levels of molecular oxygen and restricted access to nutrients due to poor vascularization in the tissue, phenomenon known as hypoxia. Hypoxic tumors are able to regulate the expression of certain genes and signaling molecules in the microenvironment that shift it towards a more aggressive phenotype. The transcriptional landscape of the tumor favors malignant transformation of neighboring cells and their migration to distant sites. Herein, we focused on identifying key proteins that participate in the signaling crossroads between hypoxic environment and metastasis progression that remain poorly defined. To shed light on these mechanisms, we performed an integrated multi-omics analysis encompassing genomic/transcriptomic alterations of hypoxia-related genes and Buffa hypoxia scores across 17 pancarcinomas taken from the PanCancer Atlas project from The Cancer Genome Atlas consortium, protein-protein interactome network, shortest paths from hypoxia-related proteins to metastatic and angiogenic phenotypes, and drugs involved in current clinical trials to treat the metastatic disease. As results, we identified 30 hypoxia-related proteins highly involved in metastasis and angiogenesis. This set of proteins, validated with the MSK-MET Project, could represent key targets for developing therapies. The upregulation of mRNA was the most prevalent alteration in all cancer types. The highest frequencies of genomic/transcriptomic alterations and hypoxia score belonged to tumor stage 4 and positive metastatic status in all pancarcinomas. The most significantly associated signaling pathways were HIF-1, PI3K-Akt, thyroid hormone, ErbB, FoxO, mTOR, insulin, MAPK, Ras, AMPK, and VEGF. The interactome network revealed high-confidence interactions among hypoxic and metastatic proteins. The analysis of shortest paths revealed several ways to spread metastasis and angiogenesis from hypoxic proteins. Lastly, we identified 23 drugs enrolled in clinical trials focused on metastatic disease treatment. Six of them were involved in advanced-stage clinical trials: aflibercept, bevacizumab, cetuximab, erlotinib, ipatasertib, and panitumumab.

MicroRNA-204/CREB5 axis regulates vasculogenic mimicry in breast cancer cells.

BACKGROUND: Vasculogenic mimicry (VM) is characterized by formation of three-dimensional (3D) channels-like structures by tumor cells, supplying the nutrients needed for tumor growth. VM is stimulated by hypoxic tumor microenvironment, and it has been associated with increased metastasis and clinical poor outcome in cancer patients. cAMP responsive element (CRE)-binding protein 5 (CREB5) is a hypoxia-activated transcription factor involved in tumorigenesis. However, CREB5 functions in VM and if its regulated by microRNAs remains unknown in breast cancer. OBJECTIVE: We aim to study the functional relationships between VM, CREB5 and microRNA-204-5p (miR-204) in breast cancer cells. METHODS: CREB5 expression was evaluated by mining the public databases, and using RT-qPCR and Western blot assays. CREB5 expression was silenced using short-hairpin RNAs in MDA-MB-231 and MCF-7 breast cancer cells. VM formation was analyzed using matrigel-based cultures in hypoxic conditions. MiR-204 expression was restored in cancer cells by transfection of RNA mimics. Luciferase reporter assays were performed to evaluate the binding of miR-204 to 3'UTR of CREB5. RESULTS: Our data showed that CREB5 mRNA expression was upregulated in a set of breast cancer cell lines and clinical tumors, and it was positively associated with poor prognosis in lymph nodes positive and grade 3 basal breast cancer patients. Silencing of CREB5 impaired the hypoxia-induced formation of 3D channels-like structures representative of the early stages of VM in MDA-MB-231 cells. In contrast, VM formation was not observed in MCF-7 cells. Interestingly, we found that CREB5 expression was negatively regulated by miR-204 mimics in breast cancer cells. Functional analysis confirmed that miR-204 binds to CREB5 3'-UTR indicating that it's an ulterior effector. CONCLUSIONS: Our findings suggested that CREB5 could be a potential biomarker of disease progression in basal subtype of breast cancer, and that perturbations of the miR-204/CREB5 axis plays an important role in VM development in breast cancer cells.

The Role of Hypoxia-Associated Long Non-Coding RNAs in Breast Cancer.

Breast cancer is the leading cause of cancer-related deaths in women worldwide. In the United States, even with earlier diagnosis and treatment improvements, the decline in mortality has stagnated in recent years. More research is needed to provide better diagnostic, prognostic, and therapeutic tools for these patients. Long non-coding RNAs are newly described molecules that have extensive roles in breast cancer. Emerging reports have shown that there is a strong link between these RNAs and the hypoxic response of breast cancer cells, which may be an important factor for enhanced tumoral progression. In this review, we summarize the role of hypoxia-associated lncRNAs in the classic cancer hallmarks, describing their effects on the upstream and downstream hypoxia signaling pathway and the use of them as diagnostic and prognostic tools.

p53 knock-down and hypoxia affects glutathione peroxidase 4 antioxidant response in hepatopancreas of the white shrimp Litopenaeus vannamei.

Hypoxia (<2 mg O2/L) is one of the main environmental stressors that affects aquatic organisms, including the white shrimp (Litopenaeus vannamei). During hypoxia, reactive oxygen species (ROS) accumulation induces oxidative stress and damage to biomolecules. Redox state and ROS overproduction are modulated by the antioxidant system that is composed of several antioxidant enzymes, proteins, and other small compounds. Glutathione peroxidase 4 (GPx4) has emerged as an important antioxidant enzyme with cytoprotective roles. In vertebrates, antioxidant and pro-oxidant stress responses are regulated by several factors, including the p53 protein. However, little is known about GPx4 responses in crustaceans and the regulation by p53. Herein we analyzed and characterized the L. vannamei GPx4 and evaluated the responses to hypoxia and p53 knock-down. We found a unique GPx4 gene that produces five transcript variants (TVs) and only two protein isoforms with distinct cellular localization. GPx4 expression in hepatopancreas during hypoxia and p53 knock-down changed during short and long-term hypoxia, suggesting that GPx4 may be a sensitive indicator of antioxidant imbalance during stress. Knock-down of p53 induced a reduction in GPx4 expression, indicating that p53 modulates GPx4 responses during stress. This agrees with our findings of putative consensus sequences for p53 in the GPx4 gene promoter by in silico analysis. Also, the antioxidant response was effective in preventing major protein damage during hypoxia since no changes were detected in carbonylated proteins content in hepatopancreas during hypoxia. Conversely, p53 knock-down produced significant changes in carbonylated proteins.

The bifunctional 6-phosphofructokinase-2/fructose-2,6-bisphosphatase from the shrimp Litopenaeus vannamei: Molecular characterization and down-regulation of expression in response to severe hypoxia.

Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.

HIF-1 is involved in the regulation of expression of metallothionein and apoptosis incidence in different oxygen conditions in the white shrimp Litopenaeus vannamei.

The white shrimp Litopenaeus vannamei is exposed to hypoxic conditions in natural habitats and in shrimp farms. Hypoxia can retard growth, development and affect survival in shrimp. The hypoxia-inducible factor 1 (HIF-1) regulates many genes involved in glucose metabolism, antioxidant proteins, including metallothionein (MT) and apoptosis. In previous studies we found that the L. vannamei MT gene expression changed during hypoxia, and MT silencing altered cell apoptosis; in this study we investigated whether the silencing of HIF-1 affected MT expression and apoptosis. Double-stranded RNA (dsRNA) was used to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia plus reoxygenation. Expression of HIF-1α, HIF-1ß and MT, and apoptosis in hemocytes or caspase-3 expression in gills, were measured at 0, 3, 24 and 48 h of hypoxia and hypoxia followed by 1 h of reoxygenation. The results showed that hemocytes HIF-1α expression was induced during hypoxia and reoxygenation at 3 h, while HIF-1ß decreased at 24 and 48 h. In normoxia, HIF-1 silencing in hemocytes increased apoptosis at 3 h and decreased at 48 h; while in gills, caspase-3 increased at 3, 24 and 48 h. In hypoxia, HIF-1 silencing decreased apoptosis in hemocytes at 3 h, but caspase-3 increased in gills. During reoxygenation, apoptosis in hemocytes and caspase-3 in gills increased. During normoxia in hemocytes, silencing of HIF-1 decreased MT expression, but in gills, MT increased. During hypoxia and reoxygenation, silencing induced MT in hemocytes and gills. These results indicate HIF-1 differential participation in MT expression regulation and apoptosis during different oxygen conditions.