Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids.

The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development.

Pharmacological induction of the hypoxia response pathway in Huh7 hepatoma cells limits proliferation but increases resilience under metabolic stress.

The hypoxia response pathway enables adaptation to oxygen deprivation. It is mediated by hypoxia-inducible factors (HIF), which promote metabolic reprogramming, erythropoiesis, angiogenesis and tissue remodeling. This led to the successful development of HIF-inducing drugs for treating anemia and some of these molecules are now in clinic. However, elevated levels of HIFs are frequently associated with tumor growth, poor prognosis, and drug resistance in various cancers, including hepatocellular carcinoma (HCC). Consequently, there are concerns regarding the recommendation of HIF-inducing drugs in certain clinical situations. Here, we analyzed the effects of two HIF-inducing drugs, Molidustat and Roxadustat, in the well-characterized HCC cell line Huh7. These drugs increased HIF-1α and HIF-2α protein levels which both participate in inducing hypoxia response genes such as BNIP3, SERPINE1, LDHA or EPO. Combined transcriptomics, proteomics and metabolomics showed that Molidustat increased the expression of glycolytic enzymes, while the mitochondrial network was fragmented and cellular respiration decreased. This metabolic remodeling was associated with a reduced proliferation and a lower demand for pyrimidine supply, but an increased ability of cells to convert pyruvate to lactate. This was accompanied by a higher resistance to the inhibition of mitochondrial respiration by antimycin A, a phenotype confirmed in Roxadustat-treated Huh7 cells and Molidustat-treated hepatoblastoma cells (Huh6 and HepG2). Overall, this study shows that HIF-inducing drugs increase the metabolic resilience of liver cancer cells to metabolic stressors, arguing for careful monitoring of patients treated with HIF-inducing drugs, especially when they are at risk of liver cancer.

Hypoxic regulation of hypoxia inducible factor 1 alpha via antisense transcription.

Impaired oxygen homeostasis is a frequently encountered pathophysiological factor in multiple complex diseases, including cardiovascular disease and cancer. While the canonical hypoxia response pathway is well characterized, less is known about the role of noncoding RNAs in this process. Here, we investigated the nascent and steady-state noncoding transcriptional responses in endothelial cells and their potential roles in regulating the hypoxic response. Notably, we identify a novel antisense long noncoding RNA that convergently overlaps the majority of the hypoxia inducible factor 1 alpha (HIF1A) locus, which is expressed across several cell types and elevated in atherosclerotic lesions. The antisense (HIF1A-AS) is produced as a stable, unspliced, and polyadenylated nuclear retained transcript. HIF1A-AS is highly induced in hypoxia by both HIF1A and HIF2A and exhibits anticorrelation with the coding HIF1A transcript and protein expression. We further characterized this functional relationship by CRISPR-mediated bimodal perturbation of the HIF1A-AS promoter. We provide evidence that HIF1A-AS represses the expression of HIF1a in cis by repressing transcriptional elongation and deposition of H3K4me3, and that this mechanism is dependent on the act of antisense transcription itself. Overall, our results indicate a critical regulatory role of antisense mediated transcription in regulation of HIF1A expression and cellular response to hypoxia.

FSH preserves the viability of hypoxic granulosa cells via activating the HIF-1α-GAS6-Axl-Akt pathway.

The developmental fate of ovarian follicles is primarily determined by the survival status (proliferation or apoptosis) of granulosa cells (GCs). Owing to the avascular environment within follicles, GCs are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH) has been reported to improve GCs survival by governing hypoxia-inducible factor-1α (HIF-1α)-dependent hypoxia response, but the underlying mechanisms remain poorly understood. Growth arrest-specific gene 6 (GAS6) is a secreted ligand of tyrosine kinase receptors, and has been documented to facilitate tumor growth. Here, we showed that the level of GAS6 was markedly increased in mouse ovarian GCs after the injection of FSH. Specifically, FSH-induced GAS6 expression was accompanied by HIF-1α accumulation under conditions of hypoxia both in vivo and in vitro, whereas inhibition of HIF-1α with small interfering RNAs/antagonist repressed both expression and secretion of GAS6. As such, Luciferase reporter assay and chromatin immunoprecipitation assay showed that HIF-1α directly bound to a hypoxia response element site within the Gas6 promoter and contributed to the regulation of GAS6 expression in response to FSH. Notably, blockage of GAS6 and/or its receptor Axl abrogated the pro-survival effects of FSH under hypoxia. Moreover, phosphorylation of Axl by GAS6 is required for FSH-mediated Akt activation and the resultant pro-survival phenotypes. Finally, the in vitro findings were verified in vivo, which showed that FSH-induced proliferative and antiapoptotic effects in ovarian GCs were diminished after blocking GAS6/Axl using HIF-1α antagonist. These findings highlight a novel function of FSH in preserving GCs viability against hypoxic stress by activating the HIF-1a-GAS6-Axl-Akt pathway.

Loss of the tumour suppressor LKB1/STK11 uncovers a leptin-mediated sensitivity mechanism to mitochondrial uncouplers for targeted cancer therapy.

Non-small cell lung cancer (NSCLC) constitutes one of the deadliest and most common malignancies. The LKB1/STK11 tumour suppressor is mutated in ∼ 30% of NSCLCs, typically lung adenocarcinomas (LUAD). We implemented zebrafish and human lung organoids as synergistic platforms to pre-clinically screen for metabolic compounds selectively targeting LKB1-deficient tumours. Interestingly, two kinase inhibitors, Piceatannol and Tyrphostin 23, appeared to exert synthetic lethality with LKB1 mutations. Although LKB1 loss alone accelerates energy expenditure, unexpectedly we find that it additionally alters regulation of the key energy homeostasis maintenance player leptin (LEP), further increasing the energetic burden and exposing a vulnerable point; acquired sensitivity to the identified compounds. We show that compound treatment stabilises Hypoxia-inducible factor 1-alpha (HIF1A) by antagonising Von Hippel-Lindau (VHL)-mediated HIF1A ubiquitination, driving LEP hyperactivation. Importantly, we demonstrate that sensitivity to piceatannol/tyrphostin 23 epistatically relies on a HIF1A-LEP-Uncoupling Protein 2 (UCP2) signaling axis lowering cellular energy beyond survival, in already challenged LKB1-deficient cells. Thus, we uncover a pivotal metabolic vulnerability of LKB1-deficient tumours, which may be therapeutically exploited using our identified compounds as mitochondrial uncouplers.

HIF1A transcriptional regulation of COX4I2 impacts angiogenesis in pheochromocytoma.

BACKGROUND: Pheochromocytoma (PCC) is a rare neuroendocrine tumor. Angiogenesis is primary contributing factor for tumorigenesis. Cytochrome c oxidase 4I2 (COX4I2) has been confirmed to take part in the progression of cancer. Hypoxia-inducible factor 1A (HIF1A) is the main regulatory factor for the steady-state response of hypoxia, involved in metabolism and angiogenesis. In this study, we intended to explore the functions of COX4I2 in PCC and the effect mechanism between HIF1A and COX4I2. MATERIALS AND METHODS: The RNA-sequencing and immunohistochemistry tested COX4I2 expression in highly vascular PCC. Small interfering RNA (siRNA) was used to reduce the mRNA expression of COX4I2, and a small molecule inhibitor was utilized to reduce the protein expression of HIF1A. Culturing cells in 1% O2environment was performed to activate HIF1A. Western blot was applied to quantify the expression of target genes at the protein levels. The supernatant from PCC cells and fibroblasts acted as the conditioned medium. We conducted the tube formation and transwell assays in human vascular endothelial cells (HUVECs) to determine angiogenesis, the binding of COX4I2 promoter and HIF1A was evaluated by the dual luciferase reporter assay. RESULTS: COX4I2 had been rigorously shown to be overexpressed in highly vascular PCC. Knockdown of COX4I2 in PCC cells (MPC) did not significantly impact angiogenesis, while knockdown of COX4I2 in fibroblast (3T3) notably inhibited angiogenesis. RNA sequencing suggested that the expression of 11 vascular markers, such as CD34 and angiogenesis associated pathways in 3T3, decreased with knockdown of COX4I2. HIF1A had been shown to enhance the mRNA expression of COX4I2 through transcriptional regulation. Activation and inhibition of HIF1A resulted in upregulation and downregulation of COX4I2, respectively. The HIF1A inhibitor demonstrated a reduction in angiogenesis. CONCLUSION: COX4I2 is overexpressed in highly vascular PCC and contributes to angiogenesis in fibroblasts. Mechanistically, HIF1A transcriptional regulation enhances COX4I2 and its effects on angiogenesis in PCC. COX4I2 might serve as a vascular marker and represent a potential target for vascular therapy.

Liensinine inhibits progression of intrahepatic cholangiocarcinoma by regulating TGF-ß1 /P-smad3 signaling through HIF-1a.

Intrahepatic cholangiocarcinoma (ICC) is a high-grade malignant digestive system tumor with an insidious onset and unfavorable prognosis. Liensinine, a small molecule derived from plants, has been proven to have significant tumor suppressor activity in other cancers. However, there are no reports on whether liensinine can inhibit the proliferation or metastasis of ICC. This study aimed to explore the tumor-suppressive activity of liensinine in ICC and its underlying mechanisms. The phenotypic changes in ICC cells were monitored in vitro using cell function tests. Western blot and immunofluorescence analyses verified the efficacy of liensinine. Tumor-bearing nude mice were used to explore the effect of liensinine on tumors and its toxicity and side effects in vivo. Liensinine suppressed ICC cell proliferation and arrested the cell cycle at the G1 phase. The epithelial-mesenchymal transition (EMT) of ICC cells was also inhibited, thereby restraining their invasion and migration of tumor cells. In addition, this study found that the potential mechanism of liensinine inhibiting EMT may be via suppression of the TGF-ß1/P-smad3 signaling pathway through hypoxia-inducible factor 1 alpha (HIF-1a). In vivo experiments showed that liensinine inhibited the growth of Hucc-T1 transplanted tumors in nude mice. Liensinine restrained the proliferation of ICC cells and suppressed EMT in ICC via the HIF-1a-mediated TGF-ß1/P-smad3 signaling pathway.

Mitochondrial metabolism in neural stem cells and implications for neurodevelopmental and neurodegenerative diseases.

Mitochondria are cytoplasmic organelles having a fundamental role in the regulation of neural stem cell (NSC) fate during neural development and maintenance.During embryonic and adult neurogenesis, NSCs undergo a metabolic switch from glycolytic to oxidative phosphorylation with a rise in mitochondrial DNA (mtDNA) content, changes in mitochondria shape and size, and a physiological augmentation of mitochondrial reactive oxygen species which together drive NSCs to proliferate and differentiate. Genetic and epigenetic modifications of proteins involved in cellular differentiation (Mechanistic Target of Rapamycin), proliferation (Wingless-type), and hypoxia (Mitogen-activated protein kinase)-and all connected by the common key regulatory factor Hypoxia Inducible Factor-1A-are deemed to be responsible for the metabolic shift and, consequently, NSC fate in physiological and pathological conditions.Both primary mitochondrial dysfunction due to mutations in nuclear DNA or mtDNA or secondary mitochondrial dysfunction in oxidative phosphorylation (OXPHOS) metabolism, mitochondrial dynamics, and organelle interplay pathways can contribute to the development of neurodevelopmental or progressive neurodegenerative disorders.This review analyses the physiology and pathology of neural development starting from the available in vitro and in vivo models and highlights the current knowledge concerning key mitochondrial pathways involved in this process.

Endothelial cell-released extracellular vesicles trigger pyroptosis and vascular inflammation to induce atherosclerosis through the delivery of HIF1A-AS2.

Extracellular vesicles (EVs) possess great potential in the modulation of cardiovascular diseases. Our current work intended to assay the clinical significance of endothelial cell (EC)-derived EVs in atherosclerosis (AS). Expression of HIF1A-AS2, miR-455-5p, and ESRRG in plasma from AS patients and mice and EVs from ox-LDL-treated ECs was measured. Interactions among HIF1A-AS2, miR-455-5p, ESRRG, and NLRP3 were analyzed. Next, EVs were co-cultured with ECs, and ectopic expression and depletion experimentations of HIF1A-AS2, miR-455-5p, ESRRG, and/or NLRP3 were carried out to assay their roles in pyroptosis and inflammation of ECs in AS. At last, the effects of HIF1A-AS2 shuttled by EC-derived EVs on EC pyroptosis and vascular inflammation in AS were verified in vivo. HIF1A-AS2 and ESRRG were highly expressed, while miR-455-5p was poorly expressed in AS. HIF1A-AS2 could sponge miR-455-5p to elevate the expression of ESRRG and NLRP3. Both in vitro and in vivo experiments revealed that ECs-derived EVs carrying HIF1A-AS2 induced the pyroptosis and vascular inflammation of ECs to promote the progression of AS by sponging miR-455-5p via ESRRG/NLRP3. HIF1A-AS2 shuttled by ECs-derived EVs can accelerate the progression of AS by downregulating miR-455-5p and upregulating ESRRG and NLRP3.

MiR-18a affects hypoxia induced glucose metabolism transition in HT22 hippocampal neuronal cell line through the Hif1a gene.

Hypoxia can cause a variety of diseases, including ischemic stroke and neurodegenerative diseases. Within a certain range of partial pressure of oxygen, cells can respond to changes in oxygen. Changes in oxygen concentration beyond a threshold will cause damage or even necrosis of tissues and organs, especially for the central nervous system. Therefore, it is very important to find appropriate measures to alleviate damage. MiRNAs can participate in the regulation of hypoxic responses in various types of cells. MiRNAs are involved in regulating hypoxic responses in many types of tissues by activating the hypoxia-inducible factor (HIF) to affect angiogenesis, glycolysis and other biological processes. By analyzing differentially expressed miRNAs in hypoxia and hypoxia-related studies, as well as the HT22 neuronal cell line under hypoxic stress, we found that the expression of miR-18a was changed in these models. MiR-18a could regulate glucose metabolism in HT22 cells under hypoxic stress by directly regulating the 3'UTR of the Hif1a gene. As a small molecule, miRNAs are easy to be designed into small nucleic acid drugs, so this study can provide a theoretical basis for the research and treatment of nervous system diseases caused by hypoxia.

Examining the relationship between genetic polymorphisms (BDKRB2, GNB3, HIF1A, MCT1, NOS3) and endurance athlete status.

PURPOSE: Genetic factors are important in terms of athletic performance. Recent studies to determine the relationship between the genes that lead to physiological responses have attracted attention. In this respect, this meta-analysis study was designed to examine the relationship between genetic polymorphism (BDKRB2 rs5810761, GNB3 rs5443, HIF1A rs11549565, MCT1 rs1049434, NOS3 rs2070744) and endurance athlete's status. METHODS: The search included studies published from 2009 to 2022. To determine the relevant studies, Pubmed, Web of Science databases were systematically scanned. Only case-control studies were included in the meta-analysis. To determine the relevant studies, Pubmed, Web of Science databases were systematically scanned, and a total of 31 studies met the criteria for inclusion in the meta-analysis. Relevant data from the included studies were collected and analyzed using a random effects or fixed effects model. The effect size was calculated as the odds ratio or a risk ratio the corresponding 95% confidence intervals. RESULTS: According to the results of the analysis, BDKRB2 rs5810761 + 9 allele, and NOS3 rs2070744 T allele were significantly more prevalent in endurance athletes (p < 0.05). Genotype distributions of BDKRB2 rs5810761, MCT1 rs1049434, and NOS3 rs2070744 showed significant differences in the dominant model (p < 0.05). However, no significant association was found between endurance athlete status and GNB3 rs5443 and HIF1A rs11549465 polymorphisms. CONCLUSION: These results show that some gene polymorphisms play an important role in endurance athlete status and suggest that having a specific genetic basis may also confer a physiological advantage for performance.

The Relation of VEGFA, VEGFR2, VEGI, and HIF1A Genetic Variants and Their Serum Protein Levels with Breast Cancer in Egyptian Patients.

Breast cancer is the most common type of cancer in Egyptian females. Polymorphisms in the angiogenesis pathway have been implicated previously in cancer risk and prognosis. The aim of the current study was to determine whether certain polymorphisms in the genes of vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial growth inhibitor (VEGI), and hypoxia-inducible factor-1α (HIF1A) associated with breast cancer development. The study included 154 breast cancer patients and 132 apparently healthy age-matched females as a control group. VEGFA rs25648 genotyping was performed using (ARMS) PCR technique; while VEGFR2 rs2071559, VEGI rs6478106, and HIF-1α rs11549465 were genotyped by the PCR-RFLP method. Serum levels of VEGF, VEGFR2, VEGI, and HIF1A proteins in breast cancer patients and controls were measured by ELISA. There was a significant association between the VEGFA rs25648 C allele and breast cancer risk (OR 2.5, 95% CI 1.7-3.6, p < 0.001). VEGFA rs25648 C/C genotype was statistically significantly higher in breast cancer patients vs. control (p < 0.001). Participants with the T/T and T/C VEGFR2 rs2071559 genotypes had 5.46 and 5 higher odds, respectively, of having breast cancer than those with the C/C genotype. For the VEGI rs6478106 polymorphism, there was a higher proportion of C allele in breast cancer patients vs. control (p = 0.003). Moreover, the C/C genotype of VEGI rs6478106 was statistically significantly higher in breast cancer patients vs. control (p = 0.001). There was no significant difference in genotypes and allele frequencies of HIF1A rs11549465 polymorphism between breast cancer cases and control individuals (p > 0.05). Serum levels of VEGFA, VEGI, and HIF1A were considerably greater in women with breast cancer than in the control (p < 0.001). In conclusion, the genetic variants VEGFA rs25648, VEGFR2 rs2071559, and VEGI rs6478106 revealed a significant association with increased breast cancer risk in Egyptian patients.

Understanding Hypoxia-Driven Tumorigenesis: The Interplay of HIF1A, DNA Methylation, and Prolyl Hydroxylases in Head and Neck Squamous Cell Carcinoma.

Hypoxia-inducible factor 1-alpha (HIF1A) is a key transcription factor aiding tumor cells' adaptation to hypoxia, regulated by the prolyl hydroxylase family (EGLN1-3) by directing toward degradation pathways. DNA methylation potentially influences EGLN and HIF1A levels, impacting cellular responses to hypoxia. We examined 96 HNSCC patients and three cell lines, analyzing gene expression of EGLN1-3, HIF1A, CA9, VEGF, and GLUT1 at the mRNA level and EGLN1 protein levels. Methylation levels of EGLNs and HIF1A were assessed through high-resolution melting analysis. Bioinformatics tools were employed to characterize associations between EGLN1-3 and HIF1A expression and methylation. We found significantly higher mRNA levels of EGLN3, HIF1A, GLUT1, VEGF, and CA9 (p = 0.021; p < 0.0001; p < 0.0001; p = 0.004, and p < 0.0001, respectively) genes in tumor tissues compared to normal ones and downregulation of the EGLN1 mRNA level in tumor tissues (p = 0.0013). In HNSCC patients with hypermethylation of HIF1A in normal tissue, we noted a reduction in HIF1A mRNA levels compared to tumor tissue (p = 0.04). In conclusion, the differential expression of EGLN and HIF1A genes in HNSCC tumors compared to normal tissues influences patients' overall survival, highlighting their role in tumor development. Moreover, DNA methylation could be responsible for HIF1A suppression in the normal tissues of HNSCC patients.

Hypoxia and HIFs in Ewing sarcoma: new perspectives on a multi-facetted relationship.

Hypoxia develops during the growth of solid tumors and influences tumoral activity in multiple ways. Low oxygen tension is also present in the bone microenvironment where Ewing sarcoma (EwS) - a highly aggressive pediatric cancer - mainly arises. Hypoxia inducible factor 1 subunit alpha (HIF-1-a) is the principal molecular mediator of the hypoxic response in cancer whereas EWSR1::FLI1 constitutes the oncogenic driver of EwS. Interaction of the two proteins has been shown in EwS. Although a growing body of studies investigated hypoxia and HIFs in EwS, their precise role for EwS pathophysiology is not clarified to date. This review summarizes and structures recent findings demonstrating that hypoxia and HIFs play a role in EwS at multiple levels. We propose to view hypoxia and HIFs as independent protagonists in the story of EwS and give a perspective on their potential clinical relevance as prognostic markers and therapeutic targets in EwS treatment.

Hypoxia-induced circRTN4IP1 promotes progression and glycolysis of hepatocellular carcinoma cells.

Hypoxia is one of the hallmarks of solid tumors, especially in hepatocellular carcinoma (HCC). CircRNAs are reported to be tightly connected to hypoxia and also have essential roles in cancer progression. However, many circRNAs implicated in hypoxia-mediated HCC progression are still unclear and require further exploration. In this study, a hypoxia cell model was structured by exposing cells to hypoxia conditions (1% O2) and normoxia conditions (21% O2) as a control. The effects of hypoxia and normoxia on cell viability, migration, invasion, and glycolysis were examined. The expressions of circRNARTN4IP1 under hypoxia were identified. Finally, molecular mechanisms and biological function of circRTN4IP1 were explored. We confirmed that hypoxia treatment facilitated capacities of proliferation, migration, invasion, and glycolysis in tumor cells. Hypoxia induced a significant increase expression of circRTN4IP1 in cells. Functionally, knockdown of circRTN4IP1 inhibited cell malignant progression and glycolysis under hypoxia HCC cells. Mechanistically, HIF1A targeted the promoter region of circRTN4IP1 and positively regulated the expression of circRTN4IP1. In addition, circRTN4IP1 targeted miR-532-5p/G6PC3 axis. In short, hypoxia induced activation of the HIF1A/circRTN4IP1/miR-532-5p/G6PC3 signaling axis, which promoted proliferation, migration, invasion, and glycolysis of HCC cells. This study may reveal a possible mechanism driving the progression of hypoxia HCC, so as to find potential effective candidates for targeting hypoxia microenvironment therapy.

lncRNA ANRIL accelerates wound healing in diabetic foot ulcers via modulating HIF1A/VEGFA signaling through interacting with FUS.

BACKGROUND: Diabetic foot ulcer (DFU) is a frequently diagnosed complication of diabetes, and remains a heathcare burden worldwide. However, the pathogenesis of DFU is still largely unclear. The objective of this study is to delineate the function and underlying mechanism of lncRNA antisense non coding RNA in the INK4 locus (ANRIL) in endothelial progenitor cells (EPCs) and DFU mice. METHODS: The DFU mouse model was established, and EPCs were subjected to high glucose (HG) treatment to mimic diabetes. qRT-PCR or western blot was employed to detected the expression of ANRIL, HIF1A, FUS and VEGFA. CCK-8 and Annexin V/PI staining were used to monitor cell proliferation and apoptosis. Wound healing, Transwell invasion and tube formation assays were conducted to assess cell migration, invasion and angiogenesis, respectively. The association between ANRIL and FUS was verified by RNA pull-down and RIP assays. Luciferase and ChIP assays were employed to investigate HIF1A-mediated transcriptional regulation of VEGFA and ANRIL. The histological alterations of DFU wound healing were observed by H&E and Masson staining. RESULTS: ANRIL was downregulated in peripheral blood samples of DFU patients, DFU mice and HG-treated EPCs. Mechanistically, ANRIL regulated HIFA mRNA stability via recruiting FUS. VEGFA and ANRIL were transcriptionally regulated by HIF1A. Functional experiments revealed that HG suppressed EPC proliferation, migration, invasion and tube formation, but promoted apoptosis via ANRIL/HIF1A axis. ANRIL accelerated DFU wound healing via modulating HIF1A expression in vivo. CONCLUSION: ANRIL accelerated wound healing in DFU via modulating HIF1A/VEGFA signaling in a FUS-dependent manner.

A novel hypoxia-stimulated lncRNA HIF1A-AS3 binds with YBX1 to promote ovarian cancer tumorigenesis by suppressing p21 and AJAP1 transcription.

Hypoxia is characteristic of the ovarian tumor (OC) microenvironment and profoundly affects tumorigenesis and therapeutic response. Long noncoding RNAs (lncRNAs) play various roles in tumor progression; however, the characteristics of lncRNAs in pathological responses of the OC microenvironment are not entirely understood. Through high-throughput sequencing, lncRNA expression in hypoxia (1% O2 ) and normoxia (21% O2 ) SKOV3 cells was explored and analyzed. The 5'- and 3'-rapid amplification of complementary DNA ends was used to detect the full length of the novel HIF1A-AS3 transcript. Real-time quantitative polymerase chain reaction was used to assess HIF1A-AS3 expression in OC cells and tissues. In vitro and in vivo evaluations of the biological functions of hypoxic HIF1A-AS3 were conducted. To clarify the underlying mechanisms of HIF1A-AS3 in hypoxic OC, a dual-luciferase assay, chromatin immunoprecipitation, RNA pull-down, RNA immunoprecipitation, and RNA-sequencing were used. We used high-throughput sequencing to investigate a novel lncRNA, HIF1A-AS3, as a hypoxic candidate significantly elevated in OC cells/tissues. HIF1A-AS3 was predominantly localized in the nucleus and promoted in vitro and in vivo OC growth and tumorigenesis. Hypoxia-inducible factor 1α bound to hypoxia response elements in the HIF1A-AS3 promoter region and stimulated its expression in hypoxia. Under hypoxia, HIF1A-AS3 directly integrated with Y-Box binding protein 1 and inhibited its ability to bind to the promoters of p21 and AJAP1 to repress their transcriptional activity, thereby promoting hypoxic OC progression. Our results revealed the crucial role and mechanism of the novel hypoxic HIF1A-AS3 in the oncogenesis of OC. The novel HIF1A-AS3 could be a crucial biomarker and therapeutic target for future OC treatments.

γ-Secretase fanning the fire of innate immunity.

Innate immunity is the first line of defense against pathogens, alerting the individual cell and surrounding area to respond to this potential invasion. γ-secretase is a transmembrane protease complex that plays an intricate role in nearly every stage of this innate immune response. Through regulation of pattern recognition receptors (PRR) such as TREM2 and RAGE γ-secretase can modulate pathogen recognition. γ-secretase can act on cytokine receptors such as IFNαR2 and CSF1R to dampen their signaling capacity. While γ-secretase-mediated regulated intramembrane proteolysis (RIP) can further moderate innate immune responses through downstream signaling pathways. Furthermore, γ-secretase has also been shown to be regulated by the innate immune system through cytokine signaling and γ-secretase modulatory proteins such as IFITM3 and Hif-1α. This review article gives an overview of how γ-secretase is implicated in innate immunity and the maintenance of its responses through potentially positive and negative feedback loops.

A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super-enhancers in maintaining EBV latency.

We probed the lifecycle of Epstein-Barr virus (EBV) on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from nine publicly available lymphoblastoid cell lines (LCLs). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350- LMP1hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1-α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1-α signaling, markedly induced this cluster. The second cluster, containing GP350+ LMP1hi cells, expressed EBV lytic genes. Host genes that are controlled by super-enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350+ LMP1hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV+ AKATA reduced the expression of these SE-regulated genes. Furthermore, CRISPR-mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV-associated heterogeneity among LCLs that may have functional consequence on host and viral biology.

NPAS2 promotes aerobic glycolysis and tumor growth in prostate cancer through HIF-1A signaling.

BACKGROUND: Prostate cancer (PCa), one of the common malignant tumors, is the second leading cause of cancer-related deaths in men. The circadian rhythm plays a critical role in disease. Circadian disturbances are often found in patients with tumors and enable to promote tumor development and accelerate its progression. Accumulating evidence suggests that the core clock gene NPAS2 (neuronal PAS domain-containing protein 2) has been implicated in tumors initiation and progression. However, there are few studies on the association between NPAS2 and prostate cancer. The purpose of this paper is to investigate the impact of NPAS2 on cell growth and glucose metabolism in prostate cancer. METHODS: Quantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) staining, western blot, GEO (Gene Expression Omnibus) and CCLE (Cancer Cell Line Encyclopedia) databases were used to analyze the expression of NPAS2 in human PCa tissues and various PCa cell lines. Cell proliferation was assessed using MTS, clonogenic assays, apoptotic analyses, and subcutaneous tumor formation experiments in nude mice. Glucose uptake, lactate production, cellular oxygen consumption rate and medium pH were measured to examine the effect of NPAS2 on glucose metabolism. The relation of NPAS2 and glycolytic genes was analyzed based on TCGA (The Cancer Genome Atlas) database. RESULTS: Our data showed that NPAS2 expression in prostate cancer patient tissue was elevated compared with that in normal prostate tissue. NPAS2 knockdown inhibited cell proliferation and promoted cell apoptosis in vitro and suppressed tumor growth in a nude mouse model in vivo. NPAS2 knockdown led to glucose uptake and lactate production diminished, oxygen consumption rate and pH elevated. NPAS2 increased HIF-1A (hypoxia-inducible factor-1A) expression, leading to enhanced glycolytic metabolism. There was a positive correlation with the expression of NPAS2 and glycolytic genes, these genes were upregulated with overexpression of NPAS2 while knockdown of NPAS2 led to a lower level. CONCLUSION: NPAS2 is upregulated in prostate cancer and promotes cell survival by promoting glycolysis and inhibiting oxidative phosphorylation in PCa cells.