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Background Interferon regulatory factor 2 (IRF-2) acts as an anti-oncogene in gastric cancer (GC); however, the underlying mechanism remains unknown. Methods This study determined the expression of IRF-2 in GC tissues and adjacent non-tumor tissues using immunohistochemistry (IHC) and explored the predictive value of IRF-2 for the prognoses of GC patients. Cell function and xenograft tumor growth experiments in nude mice were performed to test tumor proliferation ability, both in vitro and in vivo. Chromatin immunoprecipitation sequencing (ChIP-Seq) assay was used to verify the direct target of IRF-2. Results We found that IRF-2 expression was downregulated in GC tissues and was negatively correlated with the prognoses of GC patients. IRF-2 negatively affected GC cell proliferation both in vitro and in vivo. ChIP-Seq assay showed that IRF-2 could directly activate AMER-1 transcription and regulate the Wnt/β-catenin signaling pathway, which was validated using IHC, in both tissue microarray and xenografted tumor tissues, western blot analysis, and cell function experiments. Conclusions Increased expression of IRF-2 can inhibit tumor growth and affect the prognoses of patients by directly regulating AMER-1 transcription in GC and inhibiting the Wnt/β-catenin signaling pathway.

Interferon regulatory factor (IRF) 2 plays an important role in lipopolysaccharide (LPS)-induced acute kidney injury (AKI). In this study, we explored the effects of IRF2 on apoptosis, inflammation, and oxidative stress in AKI C57BL/6 male mouse model and HEK293 cells following LPS treatment. To determine the effect of IRF2, short hairpin RNAs in mice and small interfering RNAs in cells were used to knockdown IRF2 expression. IRF2 expression, apoptosis, and severity of inflammatory and oxidative stress in mice and cells were measured. IRF2 levels were upregulated in LPS-treated mice and cells. IRF2 knockdown suppressed the levels of creatinine, blood urea nitrogen, and kidney injury molecule 1 and decreased the renal injury score in mice. Furthermore, IRF2 knockdown inhibited apoptosis and decreased the levels of inflammatory, reactive oxygen species (ROS), and malondialdehyde (MDA), but increased superoxide dismutase (SOD) levels in mice and cells. Furthermore, we found that the Janus kinase (JAK)/ signal transducer and activator of transcription pathway activated by LPS was inhibited by knockdown of IRF2, and enhanced by IRF2 overexpression. IRF2 overexpression increased cell apoptosis, inflammation, and ROS and MDA levels, and decreased SOD levels. However, the effect of IRF2 overexpression was reversed by the JAK inhibitor tofacitinib. Knockdown of IRF2 reduced LPS-induced renal tissue injury in vivo and in vitro through anti-inflammatory and antioxidant stress effects.

The transcription factor interferon regulatory factor 2 (IRF2) translates interferon signaling to regulate T cells. In this issue of Immunity, Lukhele et al. identify IRF2 in tumor-infiltrating T cells as a sensor for extrinsic signals that drives an exhaustion program.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells. ispartof: FRONTIERS IN IMMUNOLOGY vol:13 ispartof: location:Switzerland status: published

IFN regulatory factor (IRF) 2 belongs to the IRF1 subfamily, and its functions are not yet fully understood. In this study, we showed that IRF2a was a negative regulator of the interferon (IFN) response induced by spring viremia of carp virus (SVCV). Irf2a(−/−) knockout zebrafish were less susceptible to SVCV than wild-type fish. Transcriptomic analysis reveals that differentially expressed genes (DEGs) in the irf2a(−/−) and irf2a(+/+) cells derived caudal fins were mainly involved in cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and transforming growth factor-beta (TGF-beta) signaling pathway. Interestingly, the basal expression levels of interferon stimulating genes (ISGs), including pkz, mx, apol, and stat1 were higher in the irf2a(−/−) cells than irf2a(+/+) cells, suggesting that they may contribute to the increased viral resistance of the irf2a(−/−) cells. Overexpression of IRF2a inhibited the activation of ifnφ1 and ifnφ3 induced by SVCV and poly(I:C) in the epithelioma papulosum cyprini (EPC) cells. Further, it was found that SVCV phosphoprotein (SVCV-P) could interact with IRF2a to promote IRF2a nuclear translocation and protein stability via suppressing K48-linked ubiquitination of IRF2a. Both IRF2a and SVCV-P not only destabilized STAT1a but reduced its translocation into the nucleus. Our work demonstrates that IRF2a cooperates with SVCV-P to suppress host antiviral response against viral infection in zebrafish. IMPORTANCE Interferon regulatory factors (IRFs) are central in the regulation of interferon-mediated antiviral immunity. Here, we reported that IRF2a suppressed interferon response and promoted virus replication in zebrafish. The suppressive effects were enhanced by the phosphoprotein of the spring viremia of carp virus (SVCV) via inhibition of K48-linked ubiquitination of IRF2a. IRF2a and SVCV phosphoprotein cooperated to degrade STAT1 and block its nuclear translocation. Our work demonstrated that IRFs and STATs were targeted by the virus through posttranslational modifications to repress interferon-mediated antiviral response in lower vertebrates.

BackgroundInterferon regulatory factor 2 (IRF2) is an important transcription factor, which can regulate the IFN response and plays a role in antiviral innate immunity in teleost.ResultsIn the present study, the full-length cDNA sequence of IRF2 (CcIRF2) was characterized in common carp (Cyprinus carpioL.), which encoded a protein containing a conserved DNA-binding domain (DBD) and an IRF-associated domain (IAD). Phylogenetic analysis showed thatCcIRF2 was most closely related with IRF2 ofCtenopharyngodon idella.CcIRF2 transcripts were detectable in all examined tissues, with higher expression in the gills, spleen and brain.CcIRF2 expression was upregulated in immune-related tissues of common carp upon polyinosinic:polycytidylic acid (poly (I:C)) andAeromonas hydrophilastimulation and induced by poly (I:C), lipopolysaccharide (LPS), peptidoglycan (PGN) and flagellin in the peripheral blood leucocytes (PBLs) and head kidney leukocytes (HKLs). In addition, overexpression ofCcIRF2 decreased the expression of IFN and IFN-stimulated genes (ISGs), and a dual-luciferase reporter assay revealed thatCcIRF2 could increase the activation of NF-κB.ConclusionsThese results indicate thatCcIRF2 participates in antiviral and antibacterial immune response and negatively regulates the IFN response, which provide a new insight into the regulation of IFN system in common carp, and are helpful for the prevention and control of infectious diseases in carp farming.

Background. Propofol is a widely used intravenous anesthetic drug with potential neuroprotective effect in diverse diseases of neuronal injuries such as traumatic brain injury and ischemic stroke. However, the underlying molecular mechanism remains largely unknown. Methods. Real-time qPCR, enzyme-linked immunosorbent assay, and Western blotting were used to identify the expression pattern of miR-221/222, inflammatory genes, cytokines, and IRF2. The biological roles and mechanisms of propofol in microglia activation were determined in BV2 cells and primary microglia. Bioinformatic analysis and luciferase reporter assay were used to confirm the regulatory role of miR-221/222 in Irf2 expression. Results. We found that miR-221 and miR-222 were downstream targets of propofol and were consistently upregulated in lipopolysaccharide- (LPS-) primed BV2 cells. Gain- and loss-of-function studies revealed that miR-221 and miR-222 were profoundly implicated in microglia activation. Then, interferon regulatory factor 2 (Irf2) was identified as a direct target gene of miR-221/222. IRF2 protein levels were reduced by miR-221/222 and increased by propofol treatment. Ectopic expression of IRF2 attenuated the proinflammatory roles induced by LPS in BV2 cells. More importantly, the suppressive effects of propofol on LPS-primed activation of BV2 cells or primary mouse microglia involved the inhibition of miR-221/222-IRF2 axis. Conclusions. Our study highlights the critical function of miR-221/222, which inhibited Irf2 translation, in the anti-inflammatory effects of propofol, and provides a new perspective for the molecular mechanism of propofol-mediated neuroprotective effect.

Osteosarcoma (OS) is the most frequently occurring bone cancer. Circular RNAs (circRNAs) have been shown to exert pivotal impact in modulation of gene expression, but their roles in OS are still not fully understood. In this study, we analysed the role of circ‐0000658 in OS. Thereafter, molecular techniques such as Western blot, qRT‐PCR, RNA‐binding protein immunoprecipitation and Dual‐Luciferase reporter assays were implemented to investigate the role of circ‐0000658/miR‐1227/interferon regulatory factor‐2 (IRF2) axis in OS. Eventually, the impact of circ‐0000658 on tumour growth and metastasis was observed in a xenograft mouse model. The results of this study revealed that circ‐0000658 exhibits low levels in OS tissues and cell lines. Moreover, circ‐0000658 repression promoted cell cycle, proliferation, invasion and migration but inhibited the apoptosis of OS cells. Researches have previously shown that circ‐0000658 contains a binding site for miR‐1227 and thus can abundantly sponge miR‐1227 to up‐regulate the expression of its target gene IRF2. Moreover, both inhibition of miR‐1227 and overexpression of IRF2 reversed cell proliferation and invasion, which was triggered by circ‐0000658 repression. Conclusively, circ‐0000658 modulates biological function of OS cells through the miR‐1227/IRF2 axis. Therefore, circ‐0000658 might act as a possible novel therapeutic target for the treatment of OS.

Background We reported that the pancreas of the interferon-regulatory factor (IRF) 2 knock-out (KO) mouse represents an early phase of acute pancreatitis, including defective regulatory exocytosis, intracellular activation of trypsin, and disturbance of autophagy. The significantly upregulated and downregulated genes in the IRF2 KO pancreas have been reported. The catalogue of gene transcripts included two types of calcium-binding proteins (S100 calcium binding protein G [S100g] and Annexin A10 [Anxa10]), which were highly upregulated in the IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and its disturbance is related to pancreatitis, we then evaluated the role of S100g and Anxa10 in acute pancreatitis. Method We induced cerulein-pancreatitis in wild-type mice and examined the changes in the expression of these genes by qPCR and immunohistochemistry. We constructed S100g-overexpressing or Anxa10-overexpressing AR42J cells (AR42J-S100g, AR42J-Anxa10). We examined the changes in amylase secretion, intracellular calcium ([Ca2+]i), and cell viability in these cells, when incubated with cholecystokinin (CCK). Results The expression of S100g and Anxa10 was increased in cerulean-induced pancreatitis. The acini were patchily stained for S100g and the cytosol of acini was evenly but weakly stained for Anxa10. Stimulation with 100pM CCK-8, decreased amylase secretion and inhibited the [Ca2+]i increase in AR42J-S100g cells. These effects were weak in AR42J-Anxa10 cells. Cell viability was not changed by incubation with cerulein. Conclusion In cerulean pancreatitis, the expression of S100g and Anxa10 was induced in the acini. S100g may work as a Ca2+ buffer in acute pancreatitis.

De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless (wg) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling., Description, Up-regulated neuronal Wnt transcription in adult brains is sufficient to cause progressive neurodegenerative phenotypes in vivo.

AIM: To assess the pathobiological and translational importance of whole blood transcriptomic analysis in inflammatory bowel disease (IBD). METHODS: We analyzed whole blood expression profiles from paired-end sequencing in a discovery cohort of 590 Europeans recruited across six countries in the IBD Character initiative (newly diagnosed patients with Crohn's disease [CD, n = 156], ulcerative colitis [UC, n = 167], and controls [n = 267]), exploring differential expression (DESeq2), co-expression networks (WGCNA), and transcription factor involvement (EPEE, ChEA, DoRothEA). Findings were validated by analysis of an independent replication cohort (99 CD, 100 UC, and 95 controls). In the discovery cohort, we also defined baseline expression correlates of future treatment escalation using cross-validated elastic-net and random forest modelling, along with a pragmatic ratio detection procedure. RESULTS: Disease-specific transcriptomes were defined in IBD (8697 transcripts), CD (7152), and UC (8521), with the most highly significant changes in single genes, including CD177 (log2-fold change [LFC] = 4.63, p = 4.05 × 10 -118), MCEMP1 (LFC = 2.45, p = 7.37 × 10 -109), and S100A12 (LFC = 2.31, p = 2.15 × 10 -93). Significantly over-represented pathways included IL-1 (p = 1.58 × 10 -11), IL-4, and IL-13 (p = 8.96 × 10 -9). Highly concordant results were obtained using multiple regulatory activity inference tools applied to the discovery and replication cohorts. These analyses demonstrated central roles in IBD for the transcription factors NFE2, SPI1 (PU.1), CEBPB, and IRF2, all regulators of cytokine signaling, based on a consistent signal across cohorts and transcription factor ranking methods. A number of simple transcriptome-based models were associated with the need for treatment escalation, including the binary CLEC5A/CDH2 expression ratio in UC (hazard ratio = 23.4, 95% CI 5.3-102.0). CONCLUSION: Transcriptomic analysis has allowed for a detailed characterization of IBD pathobiology, with important potential translational implications., Funding agency:Polish National Science Centre 2017/25/B/NZ5/02783

Yanyan Zhang, Yun Zhang, Aixiang Yang, Fei Xia Department of Critical Care Medicine, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, 215008, Jiangsu, People’s Republic of ChinaCorrespondence: Fei XiaDepartment of Critical Care Medicine, The Affiliated Suzhou Hospital of Nanjing Medical University, No. 242, Guangji Road, Suzhou, 215008, Jiangsu, People’s Republic of ChinaEmail feixia199107@163.comObjective: We investigated the roles and mechanisms of IRF2 in sepsis-related acute kidney injury (S-AKI) in a lipopolysaccharide (LPS)-induced HK-2 cell line and caecal ligation and puncture (CLP)-induced IRF2−/− mouse model.Methods: Quantitative real-time polymerase chain reaction assay was used to detect IRF2 in the serum of S-AKI patients and LPS-induced HK-2 cells. Cell proliferation, death, and apoptosis were analysed by CCK-8, lactate dehydrogenase release, and flow cytometry assays, respectively. The levels of interleukin (IL)-1β, IL-18, IL-6, tumour necrosis factor (TNF)-α, non-canonical inflammasomes, including caspase-4 and gasdermin-D (GSDMD), and canonical inflammasomes, such as caspase-1, NLR family pyrin domain containing 3 (NLRP3), and apoptosis-associated speck-like protein (ASC) in S-AKI cells or animal models were analysed by enzyme-linked immunosorbent assay or Western blotting.Results: IRF2 was upregulated in the serum of S-AKI patients and LPS-induced HK-2 cells. IRF2 downregulation promoted cell proliferation and inhibited cell death and apoptosis, respectively. IRF2 inhibition reduced the levels of IL-1β, IL-18, IL-6, and TNF-α in S-AKI cells and animal models. IRF2 knockdown inhibited LPS-treated HK-2 cell pyroptosis by decreasing the expression of caspase-4 and GSDMD, instead of affecting caspase-1, NLRP3, and ASC. An elevated survival rate and alleviated pathological features and scores were observed in the CLP-induced IRF2−/− animal models. IRF2 deficiency also suppressed inflammation and pyroptosis by inhibiting non-canonical inflammasomes as indicated by the decreased expression of caspase-11 and GSDMD.Conclusion: Our findings suggest that IRF2 downregulation protects against S-AKI in vitro and in vivo.Keywords: sepsis acute kidney injury, IRF2, inflammation, pyroptosis

Interferon regulatory factor (IRF) 2 is a transcription factor belonging to the IRF family, which is essential for gasdermin D (GSDMD)-induced pyroptosis. Decreasing myocardial cell pyroptosis confers protection against heart damage and cardiac dysfunction caused by myocardial infarction (MI). The aim of the present study was to investigate the involvement of IRF2 in MI and the underlying mechanism of IRF2 in pyroptosis. To mimic MI, ligation of the left anterior descending coronary artery was performed to establish an in vivo mouse model and rat cardiomyocytes H9c2 cells were cultured under hypoxic conditions to establish an in vitro model. Transthoracic echocardiography was used to assess cardiac function. Hematoxylin and eosin staining was used to observe histopathological changes in the myocardial tissue. Immunohistochemistry and western blotting were performed to detect IRF2 expression levels. TUNEL staining and flow cytometry were used to detect apoptosis in myocardial tissue and cells. Chromatin immunoprecipitation and dual luciferase reporter assay were used to verify the effect of IRF2 on GSDMD transcription. IRF2 was upregulated in MI mice. MI induced pyroptosis, as evidenced by increased GSDMD, N-terminal GSDMD (GSDMD-N), and cleaved (c-) caspase-1 levels. MI increased IL-1β and IL-18 levels. These alterations were alleviated by IRF2 silencing. Furthermore, in hypoxia-treated H9c2 cells, IRF2 silencing significantly decreased the elevated levels of IL-1β and IL-18 and pyroptosis-associated proteins, including GSDMD, GSDMD-N and c-caspase1. Moreover, in hypoxia-treated H9c2 cells, IRF2 directly bound to the GSDMD promoter to drive GSDMD transcription and promote pyroptosis and IRF2 expression may be regulated via the hypoxia inducible factor 1 signaling pathway. In conclusion, the present results demonstrated that IRF2 is a key regulator of MI by mediating pyroptosis, which triggers GSDMD activation.

Background Centromere protein N (CENP-N) has been reported to be highly expressed in malignancies, but its role and mechanism in nasopharyngeal carcinoma (NPC) are unknown. Methods Abnormal CENP-N expression from NPC microarrays of GEO database was analyzed. CENP-N expression level was confirmed in NPC tissues and cell lines. Stable CENP-N knockdown and overexpression NPC cell lines were established, and transcriptome sequencing after CENP-N knockdown was performed. In vitro and in vivo experiments were performed to test the impact of CENP-N knockdown in NPC cells. ChIP and dual luciferase reporter assays were used to verify the combination of IRF2 and CENP-N. Western blot analysis, cellular immunofluorescence, immunoprecipitation and GST pulldown assays were used to verify the combination of CENP-N and AKT. Results CENP-N was confirmed to be aberrantly highly expressed in NPC tissues and cell lines and to be associated with high 18F-FDG uptake in cancer nests and poor patient prognosis. Transcriptome sequencing after CENP-N knockdown revealed that genes with altered expression were enriched in pathways related to glucose metabolism, cell cycle regulation. CENP-N knockdown inhibited glucose metabolism, cell proliferation, cell cycling and promoted apoptosis. IRF2 is a transcription factor for CENP-N and directly promotes CENP-N expression in NPC cells. CENP-N affects the glucose metabolism, proliferation, cell cycling and apoptosis of NPC cells in vitro and in vivo through the AKT pathway. CENP-N formed a complex with AKT in NPC cells. Both an AKT inhibitor (MK-2206) and a LDHA inhibitor (GSK2837808A) blocked the effect of CENP-N overexpression on NPC cells by promoting aerobic glycolysis, proliferation, cell cycling and apoptosis resistance. Conclusions The IRF2/CENP-N/AKT axis promotes malignant biological behaviors in NPC cells by increasing aerobic glycolysis, and the IRF2/CENP-N/AKT signaling axis is expected to be a new target for NPC therapy.

Oral squamous cell carcinoma (OSCC) is a common type of malignant tumor worldwide. Claudin-7 (CLDN7) has been reported to exhibit low expression in tissues of patients with OSCC; however, the underlying mechanisms of CLDN7 remain to be elucidated. The present study aimed to investigate the effects of CLDN7 on the progression of OSCC and identify its potential regulatory mechanisms. CLDN7 and interferon regulatory factor-2 (IRF2) expression in several OSCC cell lines were detected using reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Following CLDN7 overexpression, cell proliferation, invasion and migration were determined using a Cell Counting Kit-8, colony formation, Transwell and wound healing assays, respectively. The potential binding sites of IRF2 on the CLDN7 promoter were analyzed using the PROMO and JASPAR databases, which were verified via chromatin immunoprecipitation and RT-qPCR assays. The effects of IRF2 and CLDN7 on the biological functions of OSCC cells were examined by transfection with short hairpin RNA (shRNA) against CLDN7 (sh-CLDN7), or IRF2 and CLDN7 overexpression plasmids. The results revealed that CLDN7 and IRF2 expression were significantly downregulated in OSCC cell lines, and CLDN7 overexpression reduced the proliferation, invasion and migration of OSCC cells. Additionally, IRF2 was confirmed to combine with the CLDN7 promoter. CLDN7 silencing reversed the inhibitory effects of IRF2 overexpression on the proliferation, invasion and migration of OSCC cells. Taken together, these findings demonstrated that IRF2-induced CLDN7 upregulation suppressed the proliferation, invasion and migration of OSCC cells, suggesting the possibility of CLDN7 and IRF2 as novel targets for the treatment of OSCC.

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function., Epidermal homeostasis requires long term stem cell function. Here, the authors apply transcriptional circuitry analysis based on integrated epigenomic profiling of primary human keratinocytes with high and low stem cell function to identify IRF2 as a negative regulator of stemness.

Tripartite motif‐containing 14 (TRIM14) is a mitochondrial adaptor that promotes innate immune signaling and plays important roles in antiviral defense. Expression of TRIM14 is induced by interferon (IFN)‐I. However, the mechanism by which IFN‐I induces TRIM14 production is not yet determined. In this study, we have examined the function of TRIM14 promoter and found that a GC box and an IFN‐stimulated response element (ISRE) are necessary for the basal level transcription of TRIM14. We further observed that IFN‐I activates the TRIM14 promoter through the ISRE. In particular, interferon regulatory factor (IRF)‐1 and IRF‐2 bind to the TRIM14 promoter and activate transcription of TRIM14. Moreover, knockdown of IRF‐1 reduces the stimulation of TRIM14 transcription by IFN‐α, suggesting that IRF‐1 is involved in the activation of TRIM14 by IFN‐I. IRF‐2 has little effect on IFN‐α‐induced TRIM14 transcription but is essential for the basal transcription of TRIM14.

Background Aberrant expression of microRNA-664 was involved in tumor growth and metastasis of various cancers. The specific role of miR-664 in cutaneous squamous cell carcinoma (cSCC) is yet to be elucidated. Objective The present study aimed to investigate the molecular mechanisms underpinning of cSCC development and provide translational insights for future therapeutics. Methods Human cSCC specimens were used to determine the miR-664 by in situhybridization and IRF2 by immunohistochemistry. To study the potential mechanisms in tumorigenesis, three cSCC cell lines including HSC-5, HSC-1 and A431 as well as BALB/C mouse tumor model was utilized. Results We found that miR-664 was remarkably high in cSCC patient specimens and cSCC cell lines. Overexpression of miR-664 promotes tumorigenic behaviors such as increased cell proliferation, migration and invasion capacities in vitro and enhanced tumorigenicity in xenograft mouse model. Our data further identified IRF2 as a direct downstream target of miR-664. Knockdown of IRF2 reverses pro-tumorigenesis phenotype of miR-664; whereas IRF2 over-expression inhibits miR-664 tumorigenesis in cSCC. Together, it revealed miR-664 functions as an oncogene in cSCC via suppression of IRF2. Conclusion Our data demonstrates that aberrant expression of miR-664 plays a critical role in carcinogenesis of cSCC. The discovery of novel targets such as miR-664 and IRF2 will facilitate future development of therapeutic interventions.

Innate lymphoid cells (ILCs), composed of heterogeneous populations of lymphoid cells, contribute critically to immune surveillance at mucosal surfaces. ILC subsets develop from common lymphoid progenitors through stepwise lineage specification. However, the composition and temporal regulation of the transcription factor network governing such a process remain incompletely understood. Here, we report that deletion of the transcription factor interferon regulatory factor 2 (IRF-2), known also for its importance in the maturation of conventional NK cells, resulted in an impaired generation of ILC1, ILC2 and ILC3 subsets with lymphoid tissue inducer (LTi)-like cells hardly affected. In IRF-2-deficient mice, PD-1hi ILC precursors (ILCPs) that generate these three ILCs but not LTi-like cells were present at normal frequency, while their sub-population expressing high amounts of PLZF, another marker for ILCPs, was severely reduced. Notably, these IRF-2-deficient ILCPs contained normal quantities of PLZF-encoding Zbtb16 messages, and PLZF expression in developing invariant NKT cells within the thymus was unaffected in these mutant mice. These results point to a unique, cell-type selective role for IRF-2 in ILC development, acting at a discrete step critical for the generation of functionally competent ILCPs.

UBE3A is a gene responsible for the pathogenesis of Angelman syndrome (AS), a neurodevelopmental disorder characterized by symptoms such as intellectual disability, delayed development and severe speech impairment. UBE3A encodes an E3 ubiquitin ligase, for which several targets have been identified, including synaptic molecules. Although proteolysis mainly occurs in the cytoplasm, UBE3A is localized to the cytoplasm and the nucleus. In fact, UBE3A is also known as a transcriptional regulator of the family of nuclear receptors. However, the function of UBE3A in the nucleus remains unclear. Therefore, we examined the involvement of UBE3A in transcription in the nuclei of neurons. Genome-wide transcriptome analysis revealed an enrichment of genes downstream of interferon regulatory factor (IRF) in a UBE3A-deficient AS mouse model. In vitro biochemical analyses further demonstrated that UBE3A interacted with IRF and, more importantly, that UBE3A enhanced IRF-dependent transcription. These results suggest a function for UBE3A as a transcriptional regulator of the immune system in the brain. These findings also provide informative molecular insights into the function of UBE3A in the brain and in AS pathogenesis.

Background Although interferon regulatory factor 2 (IRF2) was reported to stimulate virus replication by suppressing the type I interferon signaling pathway, because cell cycle arrest was found to promote viral replication, IRF2-regulated replication fork factor (FAM111A and RFC3) might be able to affect ZIKV replication. In this study, we aimed to investigate the function of IRF2, FAM111A and RFC3 to ZIKV replication and underlying mechanism. Methods siIRF2, siFAM111A, siRFC3 and pIRF2 in ZIKV-infected A549, 2FTGH and U5A cells were used to explore the mechanism of IRF2 to inhibit ZIKV replication. In addition, their expression was analyzed by RT-qPCR and western blots, respectively. Results In this study, we found IRF2 expression was increased in ZIKV-infected A549 cells and IRF2 inhibited ZIKV replication independent of type I IFN signaling pathway. IRF2 could activate FAM111A expression and then enhanced the host restriction effect of RFC3 to inhibit replication of ZIKV. Conclusions We speculated the type I interferon signaling pathway might not play a leading role in regulating ZIKV replication in IRF2-silenced cells. We found IRF2 was able to upregulate FAM111A expression and thus enhance the host restriction effect of RFC3 on ZIKV.

Background Long non-coding RNA growth arrest specific 5 (GAS5) is a regulator in non-small cell lung cancer (NSCLC) progression. Nonetheless, the mechanism by which GAS5 exerts its biological function in NSCLC cells remains unclear. Methods GAS5, miR-221-3p relative expression levels in NSCLC tissues and cells were examined by qPCR. After gain-of-function and loss-of-function models were established, the viability of H1299 and A549 cells were examined by CCK-8 and EdU assays. Cell migration and invasion were examined by the Transwell experiment. The binding sequence of GAS5 for miR-221-3p was confirmed by the dual-luciferase reporter gene experiment. The regulatory function of GAS5 and miR-221-3p on IRF2 was investigated by Western blot. Results GAS5 expression was down-modulated in NSCLC tissues and cell lines. GAS5 overexpression restrained the proliferation, migration and invasion of NSCLC cells, while miR-221-3p, which was targeted and negatively modulated by GAS5, worked oppositely. Restoration of miR-221-3p markedly reversed the effects of GAS5 on NSCLC cells. Additionally, GAS5 increased IRF2 expression in NSCLC cells by repressing miR-221-3p. Conclusions GAS5 blocks the progression of NSCLC partly via increasing IRF2 expression level via repressing miR-221-3p.

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a negative regulatory factor of interferon (IFN) and plays an important role in cell differentiation and innate immunity in mammals. In recent years, some irf8 homologous genes have been cloned and confirmed to take part in innate immune response in fish, but the mechanism still remains unclear. In this paper, a grass carp (Ctenopharyngodon idella) irf8 gene (Ciirf8) was cloned and characterized. The deduced protein (CiIRF8) possesses a highly conserved N-terminal DNA binding domain but a less well-conserved C-terminal IRF association domain (IAD). Ciirf8 was widely expressed in all tested tissues of grass carp and up-regulated following poly(I:C) stimulation. Ciirf8 expression was also up-regulated in CIK cells upon treatment with poly(I:C). To explore the molecular mechanism of how fish IRF8 regulates ifn1 expression, the similarities and differences of grass carp IRF8 and IRF2 were compared and contrasted. Subcellular localization analysis showed that CiIRF8 is located both in the cytoplasm and nucleus; however, CiIRF2 is only located in the nucleus. The nuclear-cytoplasmic translocation of CiIRF8 was observed in CIK cells under stimulation with poly(I:C). The interaction of CiIRF8 and CiIRF2 was further confirmed by a co-immunoprecipitation assay in the nucleus. Dual-luciferase reporter assays showed that the promoter activity of Ciifn1 was significantly inhibited by co-transfection with CiIRF2 and CiIRF8. The transcription inhibition of Ciifn1 was alleviated by competitive binding of CiIRF2 and CiIRF8 to CiIRF1. In conclusion, CiIRF8 down-regulates Ciifn1 expression via interaction with CiIRF2 in cells.

Interferon regulatory factors (IRFs) are crucial transcription factors involved in transcriptional regulation of type I interferons (IFNs) and IFN-stimulated genes (ISGs) against viral infection. In teleost fish, eleven IRFs have been found, however, understanding of their roles in the antiviral response remains limited. In the previous study, IRF1 (LcIRF1) and IRF2 (LcIRF2) genes were cloned from large yellow croaker (Larimichthys crocea). Here, we further characterized their function in the antiviral response. LcIRF1 and LcIRF2 were constitutively expressed in primary head kidney monocytes/macrophages (PKMs), lymphocytes (PKLs), granulocytes (PKGs) and large yellow croaker head kidney (LYCK) cell line, and significantly upregulated in PKMs and LYCK cells after stimulation with poly (I:C). LcIRF1 could induce promoter activities of three large yellow croaker type I IFNs, IFNc, IFNd and IFNh, while LcIRF2 could only induce those of IFNd and IFNh, and inhibit IFNc promoter activity. Correspondingly, overexpression of LcIRF1 in LYCK cells increased expression of all three IFNs (IFNc, IFNd and IFNh), while that of LcIRF2 only upregulated the expression levels of IFNd and IFNh, and inhibited expression of IFNc, although both LcIRF1and LcIRF2 induced expression of IFN-stimulated genes (ISGs), MxA, PKR and Viperin. Additionally, both LcIRF1 and LcIRF2 inhibited the Spring Viremia of Carp Virus (SVCV) replication in epithelioma papulosum cyprinid (EPC) cells, thus showing antiviral activity. Taken together, these results indicated that both LcIRF1 and LcIRF2 play positive roles in regulating the antiviral response of large yellow croaker by induction of distinct subgroups of type I IFNs.

The induction of an interferon-mediated response is the first line of defense against pathogens such as viruses. Yet, the dynamics and extent of interferon alpha (IFNα)-induced antiviral genes vary remarkably and comprise three expression clusters: early, intermediate and late. By mathematical modeling based on time-resolved quantitative data, we identified mRNA stability as well as a negative regulatory loop as key mechanisms endogenously controlling the expression dynamics of IFNα-induced antiviral genes in hepatocytes. Guided by the mathematical model, we uncovered that this regulatory loop is mediated by the transcription factor IRF2 and showed that knock-down of IRF2 results in enhanced expression of early, intermediate and late IFNα-induced antiviral genes. Co-stimulation experiments with different pro-inflammatory cytokines revealed that this amplified expression dynamics of the early, intermediate and late IFNα-induced antiviral genes can also be achieved by co-application of IFNα and interleukin1 beta (IL1β). Consistently, we found that IL1β enhances IFNα-mediated repression of viral replication. Conversely, we observed that in IL1β receptor knock-out mice replication of viruses sensitive to IFNα is increased. Thus, IL1β is capable to potentiate IFNα-induced antiviral responses and could be exploited to improve antiviral therapies., Author summary Innate immune responses contribute to the control of viral infections and the induction of interferon alpha (IFNα)-mediated antiviral responses is an important component. However, IFNα induces a multitude of antiviral response genes and the expression dynamics of these genes can be classified as early, intermediate and late. Here we show, based on a mathematical modeling approach, that mRNA stability as well as the negative regulator IRF2 control the expression dynamics of IFNα-induced antiviral genes. Knock-down of IRF2 resulted in the amplified IFNα-mediated induction of the antiviral genes and this amplified expression of antiviral genes could be functionally mimicked by co-stimulation with IFNα and IL1β. We observed that co-stimulation with IFNα and IL1β enhanced the repression of virus replication and that knock-out of the IL1 receptor in mice resulted in increased replication of a virus sensitive to IFNα. In sum, our studies identified IL1β as an important amplifier of IFNα-induced antiviral responses.

The physiological stresses that diminish tissue stem-cell characteristics remain largely unknown. We previously reported that type I interferon (IFN), which is essential for host antiviral responses, is a physiological stressor for hematopoietic stem cells (HSCs) and small intestinal stem cells (ISCs) and that interferon regulatory factor-2 (IRF2), which attenuates IFN signaling, maintains their stemness. Here, using a dextran sodium sulfate (DSS)-induced colitis model, we explore the role of IRF2 in maintaining colonic epithelial stem cells (CoSCs). In mice with a conditional Irf2 deletion in the intestinal epithelium (hereafter Irf2ΔIEC mice), both the number and the organoid-forming potential of CoSCs were markedly reduced. Consistent with this finding, the ability of Irf2ΔIEC mice to regenerate colon epithelium after inducing colitis was severely impaired, independently of microbial dysbiosis. Mechanistically, CoSCs differentiated prematurely into transit-amplifying (TA) cells in Irf2ΔIEC mice, which might explain their low CoSC counts. A similar phenotype was induced in wild-type mice by repeated injections of low doses of poly(I:C), which induces type I IFN. Collectively, we demonstrated that chronic IFN signaling physiologically stresses CoSCs. This study provides new insight into the development of colitis and molecular mechanisms that maintain functional CoSCs throughout life.

The objective response rate of immune checkpoint blockade (ICB) in hepatocellular carcinoma (HCC) with anti PD-L1/PD-1 therapy is low. Discovering the signaling pathways regulating PD-L1 might help to improve ICB response rates. Here, we investigate transcription factors IRF-1 and IRF-2 signaling pathways regulating PD-L1 in HCC cells. In vivo studies show that IRF-1 and PD-L1 mRNA expression in human HCC tumors are significantly repressed compared with noncancerous background liver. IRF-1, IRF-2, and PD-L1 mRNA expression correlated positively in HCC tumors. Increased IRF-1 mRNA expression was observed in patients with well-differentiated or early stage HCC tumors. In vitro studies show that IFN-γ induces PD-L1 mRNA and protein expression through upregulation of IRF-1 in mouse and human HCC cells. IRF-1, IRF-2, and PD-L1 mRNA expression is upregulated in murine HCC by co-culture with effector T cells from spleen cells incubated with anti-CD3/CD28 antibodies. IRF-2 over-expression down-regulates IFN-γ induced PD-L1 promoter activity and protein levels in a dose-dependent manner. We identify two IRF-1 response elements (IRE1/IRE2) in the upstream 5'-flanking region of the CD274 (PD-L1) gene promoter. Site-directed mutagenesis shows both IRE1 and IRE2 are functional in transfection promoter assays. IRF-1 traditionally functions as tumor suppressor gene. However, these novel findings show a complex role for IRF-1 which upregulates PD-L1 in the inflammatory tumor microenvironment. IRF-1 antagonizes IRF-2 for binding to the IRE promoter element in PD-L1 which gives new insight to the regulation of PD-L1/PD-1 pathways in HCC ICB therapy.

Chemoresistance is the leading cause of recurrence in non-small cell lung cancer (NSCLC). The long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) inhibits the tumorigenesis of various cancers. However, the regulatory function of CASC2 on the chemoresistance of NSCLC remains unclear.The levels of CASC2 and miR-18a in cisplatin (DDP)-resistant NSCLC tissues and cell lines were evaluated by quantitative Polymerase Chain Reaction (qPCR). The role of low CASC2 levels on overall survival in patients with NSCLC was tested using the log-rank test. The Chi-squared test was used to assess the relation between CASC2 expression and clinicopathological features of NSCLC patients. Cell Counting Kit-8 (CCK-8) assays tested the cell proliferation of cisplatin-resistant NSCLC cells (H226/DDP and A549/DDP). The underlying regulatory mechanism between CASC2 and miR-18a or miR-18a and interferon regulatory factor 2 (IRF-2) was predicted by bioinformatics and verified by a Dual-Luciferase reporter assay, RNA transfection, qPCR, and Western blotting. Chromatin immunoprecipitation (ChIP) assay was done to exam the relation between E74 like factor 1 (ELF1) and CASC2 gene. Mice xenografts were applied to exam the function of CASC2 on chemosensitivity of cisplatin of NSCLC cells in vivo.Low CASC2 expression is more likely to present in patients with advanced TNM stage (IV), cisplatin-resistance, and poor overall survival. The expression of CASC2 sharply decreased in cisplatin-resistant NSCLC tissues and cell lines (H226/DDP and A549/DDP). CASC2 overexpression strongly inhibited proliferation, migration, and invasion of cisplatin-resistant NSCLC cells (H226/DDP and A549/DDP) in vitro and inhibited tumor growth in vivo. Besides, CASC2 repressed miR-18a function by binding to the complementary sites of miR-18a as competing endogenous RNAs (ceRNAs). MiR-18a released by the declining expression of CASC2 reduced the protein concentration of IRF-2 in NSCLC cells. Furthermore, the transcription factor ELF1 was found to be promotor of CASC2 and increased its levels in cisplatin-resistant NSCLC cells.IRF-2 expression mediated by the ELF1/CASC2/miR-18a axis is markedly associated with the proliferation, migration, and invasion of cisplatin-resistant NSCLC, resulting in inferior survival. These findings suggest that this regulatory axis may serve as a novel therapeutic target in NSCLC.

Intestinal stem cells (ISCs) are located at the crypt base and fine-tune the balance of their self-renewal and differentiation

Background and aims Major vault protein (MVP) is up-regulated during infections with hepatitis B virus (HBV) and hepatitis C virus (HCV). Here, we found that MVP deficiency inhibited hepatocellular carcinoma (HCC) development induced by diethylnitrosamine, hepatitis B X protein, and HCV core. Approach and results Forced MVP expression was sufficient to induce HCC in mice. Mechanistic studies demonstrate that the ubiquitin ligase human double minute 2 (HDM2) forms mutual exclusive complexes either with interferon regulatory factor 2 (IRF2) or with p53. In the presence of MVP, HDM2 is liberated from IRF2, leading to the ubiquitination of the tumor suppressor p53. Mouse xenograft models showed that HBV and HCV promote carcinogenesis through MVP induction, resulting in a loss of p53 mediated by HDM2. Analyses of clinical samples from chronic hepatitis B, liver cirrhosis, and HCC revealed that MVP up-regulation correlates with several hallmarks of malignancy and associates with poor overall survival. Conclusions Taken together, through the sequestration of IRF2, MVP promotes an HDM2-dependent loss of p53 that promotes HCC development.

Fetal growth restriction (FGR) is a serious pregnancy complication associated with increased perinatal mortality and morbidity. It may lead to neurodevelopmental impairment and adulthood onset disorders. Recently, long noncoding RNAs (lncRNAs) were found to be associated with the pathogenesis of FGR. Here we report that the lncRNAH19 is significantly decreased in placentae from pregnancies with FGR. Downregulation of H19 leads to reduced proliferation and invasion of extravillous trophoblast cells. This is identified with reduced trophoblast invasion, which has been discovered in FGR. Autophagy is exaggerated in FGR. Downregulation of H19 promotes autophagy via the PI3K/AKT/mTOR and MAPK/ERK/mTOR pathways of extravillous trophoblast cells in FGR. We also found that the expression level of microRNAs miR‐18a‐5p was negatively correlated with that of H19. H19 can act as an endogenous sponge by directly binding to miR‐18a‐5p, which targets IRF2. The expression of miR‐18a‐5p was upregulated, but IRF2 expression was downregulated after the H19 knockdown. In conclusion, our study revealed that H19 downexpressed could inhibit proliferation and invasion, and promote autophagy by targeting miR‐18a‐5pin HTR8 and JEG3 cells. We propose that aberrant regulation of H19/miR‐18a‐5p‐mediated regulatory pathway may contribute to the molecular mechanism of FGR. We indicated that H19 may be a potential predictive, diagnostic, and therapeutic modality for FGR.

Long noncoding RNAs (lncRNAs), once thought to be nonfunctional, have recently been shown to participate in the multilevel regulation of transcriptional, posttranscriptional and epigenetic modifications and to play important roles in various biological processes, including immune responses. However, the expression and roles of lncRNAs in invertebrates, especially nonmodel organisms, remain poorly understood. In this study, by comparing a transcriptome to the PfIRF-2 genomic structure, we identified lncIRF-2 in the PfIRF-2 genomic intron. The results of the RNA interference (RNAi) and the nucleus grafting experiments indicated that PfIRF-2 might have a negative regulatory effect on lncIRF-2, and PfIRF-2 and lncIRF-2 may have a positive regulatory effect on PfIL-17. Additionally, lncIRF-2, PfIRF-2 and PfIL-17 were involved in responses to the nucleus graft. These results will enhance the knowledge of lncIRF-2, IRF-2, and IL-17 functions in both pearl oysters and other invertebrates.

Transforming growth factor β1 (TGF-β1) plays a promoting role in tumor growth via a mechanism associated with hyperactive Smad3 and suppressed Smad7 signaling in the tumor microenvironment. We report that retrieving the balance between Smad3 and Smad7 signaling with asiatic acid (AA, a Smad7 inducer) and naringenin (NG, a Smad3 inhibitor) effectively inhibited tumor progression in mouse models of invasive melanoma (B16F10) and lung carcinoma (LLC) by promoting natural killer (NK) cell development and cytotoxicity against cancer. Mechanistically, we found that Smad3 physically bound Id2 and IRF2 to suppress NK cell production and NK cell-mediated cytotoxicity against cancer. Treatment with AA and NG greatly inhibited Smad3 translation and phosphorylation while it restored Smad7 expression, and, therefore, it largely promoted NK cell differentiation, maturation, and cytotoxicity against cancer via Id2/IRF2-associated mechanisms. In contrast, silencing Id2 or IRF2 blunted the protective effects of AA and NG on NK cell-dependent anti-cancer activities. Thus, treatment with AA and NG produced an additive effect on inactivating TGF-β1/Smad3 signaling, and, therefore, it suppressed melanoma and lung carcinoma growth by promoting NK cell immunity against cancer via a mechanism associated with Id2 and IRF2.

Backgrounds IFN regulatory factor (IRF)-2 is one of the potential susceptibility genes for psoriasis, but how this gene influences psoriasis pathogenesis is unclear. Topical application of imiquimod (IMQ), a TLR7 ligand, induces psoriasis-like skin lesions in mice. Objective The aim of this study was to investigate whether IRF-2 gene status would influence severity of skin disease in IMQ-treated mice. Methods Imiquimod-induced psoriasis-like skin inflammation was assessed by clinical findings, histology, and cytokine expression. The effects of imiquimod or IFN on peritoneal macrophages were analyzed in vitro. Results IMQ-induced skin inflammation assessed by clinical findings and histology was more severe in IRF-2+/− mice than in wild-type mice. In inflamed skin, mRNA expression levels of tumor necrosis factor (TNF)-α, IL-12/23p40, IL-17A, and IL-22 were significantly elevated in IRF-2+/− mice compared to wild-type mice. Stimulation of peritoneal macrophages by IMQ significantly increased mRNA levels of TNF-α, IL-12/23p40, IL-23p19, IL-12p35, and IL-36. Interestingly, macrophages from IRF-2+/− mice expressed higher levels of TNF-α, IL-12/23p40, and IL-36 compared to those from wild-type mice 24 h after stimulation, while they expressed similar levels of IL-12p35 and IL-23p19. Moreover, elevated mRNA expression of inducible nitric oxide synthase was observed only in IMQ-stimulated macrophages derived from IRF-2+/− mice, which correlated with angiogenesis in IMQ-treated ears of IRF-2+/− mice. Conclusions These results suggest that IRF-2 haploinsufficiency creates heightened biologic responses to IFN-α that phenotypically lead to enhanced angiogenesis and psoriasis-like inflammation within skin.

Acute myeloid leukemia (AML) is a highly heterogeneous disease, which results in the fact that patient management has remained disappointingly uniform. Therefore, the molecular mechanism underlying AML needs to be further investigated. Here in this study, we identify the interferon-regulatory factor 2 (IRF2) as a novel regulator in human AML. We show that IRF2 knockdown inhibits growth, colony formation of OCI/AML-2, OCI/AML-3, and THP-1 cells. In addition, IRF2 knockdown induces apoptosis of AML cells by regulating the apoptotic effectors Bcl-2, Bax and Caspase 3. Further mechanism analysis shows that inositol polyphosphate-4-phosphatase, type-II (INPP4B) contributes to the effects of IRF2 on apoptosis and growth of AML cells. IRF2 binds INPP4B promoter and promotes INPP4B expression in AML cells. Restoration of the expression of INPP4B blocks the effects of IRF2 knockdown on apoptosis and colony formation in OCI/AML-2 and OCI/AML-3 cells. In conclusion, IRF2 serves as an important regulator in AML by targeting INPP4B. Therefore, IRF2 may be a potential target for AML treatment.

Interferon regulatory factors (IRFs) are transcription factors that play important roles in immune defense, stress response, hematopoietic differentiation, and cell apoptosis. IRFs of invertebrate organisms and their functions remain largely unexplored. In the present study, for the first time new IRFs (BpIRF2 and MmIRF2) were identified in the deep-sea mussel Bathymodiolus platifrons and the shallow-water mussel Modiolus modiolus. The open reading frame of BpIRF2 and MmIRF2 encoded putative proteins of 354 and 348 amino acids, respectively. Comparison and phylogenetic analysis revealed that both IRF2 proteins were new identified invertebrate IRF molecular. As transcriptional factors, both BpIRF2 and MmIRF2 could activate the interferon-stimulated response element-containing promoter and BpIRF2 could interact with itself. Moreover, both BpIRF2 and MmIRF2 were localized to the cytoplasm and nucleus. Collectively, these results demonstrated that IRF2 proteins might be crucial in the innate immunity of deep-sea and shallow-water mussels.

PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a protective protein which regulates the adaptation of cells to ER stress and virus-stimulated signaling pathways by activating PKR. In the present study, a grass carp (Ctenopharyngodon idella) PRKRA full-length cDNA (named CiPRKRA, KT891991) was cloned and identified. The full-length cDNA is comprised of a 5′ UTR (36 bp), a 3′ UTR (350 bp) and the longest ORF (882 bp) encoding a polypeptide of 293 amino acids. The deduced amino acid sequence of CiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship of CiPRKRA with other fish PRKRA, especially with Danio rerio PRKRA. qRT-PCR showed that CiPRKRA was significantly up-regulated after stimulation with tunicamycin (Tm) and Poly I:C in C. idella kidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence of CiPRKRA (1463 bp) containing one ISRE and one CARE was cloned by Tail-PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, both CiIRF2 and CiATF4 bound to CiPRKRA promoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors for CiPRKRA. Dual-luciferase reporter assays were applied to further investigate the transcriptional regulation of CiPRKRA in vivo. Recombinant plasmid of pGL3-PRKRAPro was constructed and transiently co-transfected into CIK cells with pcDNA3.1-CiIRF2 and pcDNA3.1-CiATF4, respectively. The results showed that both CiIRF2 and CiATF4 significantly decreased the luciferase activity of pGL3-PRKRAPro, suggesting that they play a negative role in CiPRKRA transcription.

Interferon regulatory factor 2 (IRF-2) is known to play a pivotal role in the development and progression of several malignancies. As a crucial member of interferon regulatory factor family, the association between the expression of IRF-2 and clinical prognostic significance has not been fully explored in colorectal cancer (CRC). The purpose of our study was to investigate the expression profile of IRF-2 in CRC and to examine its association with clinical features. The expression levels of IRF-2 in 18 paired CRC and non-cancerous colorectal tissues were measured by quantitative real-time PCR (qRT-PCR) and those in 4 paired samples by Western blotting. The results showed a significant increase in IRF-2 mRNA expression and protein expression in CRC tissues compared to those in paired normal tissues. Besides, high expression of IRF-2 was significantly associated with distant metastasis (P = 0.041) and preoperative serum CEA level (P = 0.045). Kaplan–Meier survival analysis showed that patients with high expression of IRF-2 had a significantly worse overall survival than those with low expression of IRF-2 (P = 0.006). Further multivariate analysis indicated that IRF-2 and TNM stage were independent prognostic factors for overall survival in patients with CRC. Our study primarily suggests IRF-2 as a potential prognostic biomarker in CRC.

Cancer related inflammation (CRI) plays an important role in the development of esophageal cancer (EC), and the target gene analysis shows that miR-302b potential target genes closely correlated to CRI important signaling pathways. The present study was to evaluate the inhibition of miR-302b on CRI in EC and its mechanism. We found that the expression levels of miR-302b in EC cells were lower than that in Het-1A cells, while TE11 with the lowest expression and OE33 with the highest. Inflammatory stimuli at 48 h significantly reduced expression of miR-302b in EC cells, but had no effect in Het-1A. After up-regulation of miR-302b in TE11 and down-regulation of miR-302b in OE33, it was found that miR-302b reduced CRI key transcription factors and representative cytokines. Then, over-expressed of miR-302b significantly altered potential target genes protein expressions and there was a negative correlation between miR-302b and potential target genes protein expressions (ERBB4, IRF2 and CXCR4) in EC tissues. Then reporter gene analysis revealed that miR-302b post-transcriptionally regulated expression of target genes by specific area of 3′-UTR. Transfected by target genes shRNA plasmids together could get the same effects of miR-302b on protein expression of CRI key transcription factors. Furthermore, miR-302b was able to repress tumor growth and transcription factors protein expression in vivo. These finding suggests that miR-302b inhibits key transcription factors and cytokines by targeting ERBB4, IRF2 and CXCR4, implicating its role in the inhibition of CRI in EC.

Acute kidney injury is a common and severe complication of critical illness and cardiac surgery. Despite significant attempts at developing treatments, therapeutic advances to attenuate acute kidney injury and expedite recovery have largely failed.Identifying genetic loci associated with increased risk of acute kidney injury may reveal novel pathways for therapeutic development.We conducted an exploratory genome-wide association study to identify single-nucleotide polymorphisms associated with genetic susceptibility to in-hospital acute kidney injury.We genotyped 609,508 single-nucleotide polymorphisms and performed genotype imputation in 760 acute kidney injury cases and 669 controls. We then evaluated polymorphisms that showed the strongest association with acute kidney injury in a replication patient population containing 206 cases with 1,406 controls. We observed an association between acute kidney injury and four single-nucleotide polymorphisms at two independent loci on metaanalysis of discovery and replication populations. These include rs62341639 (metaanalysis P = 2.48 × 10Our findings reveal two genetic loci that are associated with acute kidney injury. Additional studies should be conducted to functionally evaluate these loci and to identify other common genetic variants contributing to acute kidney injury.

Interferon regulatory factors (irfs) are a family of genes that encode transcription factors with important roles in regulating the expression of Type I interferons (IFNs) and other genes associated with related pathways. irfs have multitudinous functions in growth, development and regulation of oncogenesis. In this study, three irf family members (irf2, irf7, irf9) were identified and characterized in Megalobrama amblycephala at the mRNA and amino acid levels. M. amblycephala irfs share a high sequence homology with other vertebrate irfs. Constitutive expression levels of the three genes were detected (using qPCR) in all studied tissues: low to medium in kidney, gills, heart and muscle, and high in liver, spleen, intestine and blood. qPCR was also used to analyze the dynamic expression patterns of irfs in different embryonic development stages: irf2 is not activated during the embryonic development, whereas irf9 appears to play important roles around hatching and during the larval development. Transcripts of all three studied irfs were upregulated after stimulation by Aeromonas hydrophila bacterium in liver, spleen, head kidney and trunk kidney, whereas downregulation was observed in intestine and gills. The results show that these three irfs are likely to be important factors in the blunt snout bream immune system. They also provide a foundation for studying the origin and evolution of the innate immune system in the blunt snout bream.