Nuclear Factor I-A Research Articles

Spinal motor neuron development and metabolism are transcriptionally regulated by Nuclear Factor IA.

Neural circuits governing all motor behaviors in vertebrates rely on the proper development of motor neurons and their precise targeting of limb muscles. Transcription factors are essential for motor neuron development, regulating their specification, migration, and axonal targeting. While transcriptional regulation of the early stages of motor neuron specification is well-established, much less is known about the role of transcription factors in the later stages of maturation and terminal arborization. Defining the molecular mechanisms of these later stages is critical for elucidating how motor circuits are constructed. Here, we demonstrate that the transcription factor Nuclear Factor-IA (NFIA) is required for motor neuron positioning, axonal branching, and neuromuscular junction formation. Moreover, we find that NFIA is required for proper mitochondrial function and ATP production, providing a new and important link between transcription factors and metabolism during motor neuron development. Together, these findings underscore the critical role of NFIA in instructing the assembly of spinal circuits for movement.

Association of ARRDC3 and NFIA variants with bovine congestive heart failure in feedlot cattle.

Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease. A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar's test. Two independent genomic regions were identified as having the most significant association with BCHF: the arrestin domain-containing protein 3 gene ( ARRDC3), and the nuclear factor IA gene ( NFIA, mid- p-values, 1x10 -8 and 2x10 -7, respectively). Animals with two copies of risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates with either one or zero risk alleles at both genes (CI 95 = 3-17). Further, animals with two copies of risk alleles at both genes were 28-fold more likely to have BCHF than all others ( p-value = 1×10 -7, CI 95 = 4-206). A missense variant in ARRDC3 (C182Y) represents a potential functional variant since the C182 codon is conserved among all other jawed vertebrate species observed. A two-SNP test with markers in both genes showed 29% of 273 BCHF cases had homozygous risk genotypes in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This and other DNA tests may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here. Although pathogenic roles for variants in the ARRDC3 and NFIA genes are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management.

Cell-specific NFIA upregulation promotes epileptogenesis by TRPV4-mediated astrocyte reactivity

BackgroundThe astrocytes in the central nervous system (CNS) exhibit morphological and functional diversity in brain region-specific pattern. Functional alterations of reactive astrocytes are commonly present in human temporal lobe epilepsy (TLE) cases, meanwhile the neuroinflammation mediated by reactive astrocytes may advance the development of hippocampal epilepsy in animal models. Nuclear factor I-A (NFIA) may regulate astrocyte diversity in the adult brain. However, whether NFIA endows the astrocytes with regional specificity to be involved in epileptogenesis remains elusive.MethodsHere, we utilize an interference RNA targeting NFIA to explore the characteristics of NFIA expression and its role in astrocyte reactivity in a 4-aminopyridine (4-AP)-induced seizure model in vivo and in vitro. Combined with the employment of a HA-tagged plasmid overexpressing NFIA, we further investigate the precise mechanisms how NIFA facilitates epileptogenesis.Results4-AP-induced NFIA upregulation in hippocampal region is astrocyte-specific, and primarily promotes detrimental actions of reactive astrocyte. In line with this phenomenon, both NFIA and vanilloid transient receptor potential 4 (TRPV4) are upregulated in hippocampal astrocytes in human samples from the TLE surgical patients and mouse samples with intraperitoneal 4-AP. NFIA directly regulates mouse astrocytic TRPV4 expression while the quantity and the functional activity of TRPV4 are required for 4-AP-induced astrocyte reactivity and release of proinflammatory cytokines in the charge of NFIA upregulation. NFIA deficiency efficiently inhibits 4-AP-induced TRPV4 upregulation, weakens astrocytic calcium activity and specific astrocyte reactivity, thereby mitigating aberrant neuronal discharges and neuronal damage, and suppressing epileptic seizure.ConclusionsOur results uncover the critical role of NFIA in astrocyte reactivity and illustrate how epileptogenic brain injury initiates cell-specific signaling pathway to dictate the astrocyte responses.

Hsa-miR-223-3p participates in the process of anthracycline-induced cardiomyocyte damage by regulating NFIA gene.

Irreversible cardiomyopathy was caused by the therapeutic of anthracyclines in the chemotherapy of cancers. The cell apoptosis and autophagy were induced by anthracyclines in AC16 cells. MiR-223-3p ascends in anthracycline-treated AC16, but the expression of nuclear factor I-A (NFIA) was specifically down-regulated. However, the underlying molecular mechanism between NFIA and miR-223-3p is unclear now in AC16 cells. In our research, NFIA expression was dampened in AC16 cells by miR-223-3p mimics. Additionally, miR-223-3p knockdown hindered the apoptosis and autophagy in anthracycline-treated AC16. Furthermore, NFIA was predicted and verified as a miR-223-3p's downstream target and rescued the functions of miR-223-3p. These findings illustrated that miR-223-3p advances anthracycline-stimulated cardiomyocyte damage progression by targeting NFIA, implying the promising therapeutic function of miR-223-3p on cardiomyocyte damage in cancer patients.

NFIA in adipocytes reciprocally regulates mitochondrial and inflammatory gene program to improve glucose homeostasis

Adipose tissue is central to regulation of energy homeostasis. Adaptive thermogenesis, which relies on mitochondrial oxidative phosphorylation (Ox-Phos), dissipates energy to counteract obesity. On the other hand, chronic inflammation in adipose tissue is linked to type 2 diabetes and obesity. Here, we show that nuclear factor I-A (NFIA), a transcriptional regulator of brown and beige adipocytes, improves glucose homeostasis by upregulation of Ox-Phos and reciprocal downregulation of inflammation. Mice with transgenic expression of NFIA in adipocytes exhibited improved glucose tolerance and limited weight gain. NFIA up-regulates Ox-Phos and brown-fat-specific genes by enhancer activation that involves facilitated genomic binding of PPARγ. In contrast, NFIA in adipocytes, but not in macrophages, down-regulates proinflammatory cytokine genes to ameliorate adipose tissue inflammation. NFIA binds to regulatory region of the Ccl2 gene, which encodes proinflammatory cytokine MCP-1 (monocyte chemoattractant protein-1), to down-regulate its transcription. CCL2 expression was negatively correlated with NFIA expression in human adipose tissue. These results reveal the beneficial effect of NFIA on glucose and body weight homeostasis and also highlight previously unappreciated role of NFIA in suppressing adipose tissue inflammation.

SOX9/NFIA promotes human ovarian cancer metastasis through the Wnt/β-catenin signaling pathway

To our knowledge, Sex-determining Region Y box 9 (SOX9) has been in connection with a wide range of human cancers. Nevertheless, there remains uncertainty regarding SOX9's role in metastasizing ovarian cancer. In our study, SOX9 was investigated in relation to tumor metastasis in ovarian cancer as well as its potential molecular mechanisms. First, we exhibited an apparent higher expression of SOX9 in ovarian cancer tissues and cells than in normative ones, and the prognosis of patients whose SOX9 levels were high was markedly lower than that of patients whose SOX9 levels were low. Besides, highly expressed SOX9 was correlated with high grade serous carcinoma, poor tumor differentiation, high serum CA125 and lymph node metastasis. Second, SOX9 knockdown exhibited striking inhibition of the migration and invasive ability of ovarian cancer cells, whereas SOX9 overexpression had an inverse role. At the same time, SOX9 could promote ovarian cancer intraperitoneal metastasis in a nude mice in the vivo. In a similar way, SOX9 knockdown dramatically decreased the expression of nuclear factor I-A (NFIA), β-catenin as well as N-cadherin but had an increased in E-cadherin expression, as opposed to the results when SOX9 was overexpressed. Furthermore, NFIA silencing inhibited the expression of NFIA, β-catenin and N-cadherin, in the same way that E-cadherin expression was promoted. In conclusion, this study shows that SOX9 has a promotional effect on human ovarian cancer and that SOX9 promotes the metastasis of tumors by upregulating NFIA and activating on a Wnt/β-catenin signal pathway. SOX9 could be a novel focus for earlier diagnosis, therapy and prospective evaluation in ovarian cancer.

RGC-32 facilitates reactive astrocytosis by modulating the expression of axonal guidance molecules

Abstract Response Gene to Complement (RGC)-32 modulates TGF-β-induced extracellular matrix secretion and the ability of astrocytes to undergo reactive changes in vivo during experimental autoimmune encephalomyelitis (EAE). However, the molecular pathways underlying these effects are still not well understood. In this study, we investigated how lack of RGC-32 affects the transcriptomic profile and the expression of axonal guidance molecules (AGM) in astrocytes during EAE. We performed next-generation RNA sequencing on brain neonatal astrocytes isolated from wild type (WT) and RGC-32 knock-out (KO) mice, either unstimulated or stimulated with TGF-β. Results were then validated by using Real-Time PCR. Spinal cords from WT and RGC-32 KO mice with EAE (at days 0 and 14) were stained by immunohistochemistry for the astrocyte marker GFAP, AGM, and for nuclear factor IA (NFIA), a gliogenic factor and transcriptional regulator of AGM. Lack of RGC-32 had a significant impact on the transcriptomic programs normally associated with brain development whose re-expression is usually seen in reactive astrocytes. Connectivity analysis revealed that genes coding for AGM were particularly affected. We found lower transcript levels of ephrin receptor A type 7 (Epha7), plexin A1 and Slit guidance ligand 2 in RGC-32 KO astrocytes. Moreover, our results showed that NFIA and EPHA7 are expressed by astrocytes during EAE. We found a lower number of astrocytes expressing EPHA7 and NFIA in RGC-32 KO mice with acute EAE when compared with WT mice. These results suggest that RGC-32 might facilitate reactive astrogliosis during acute EAE through regulating the expression of AGM and NFIA. Veterans Administration Merit Award I01BX001458 (to HR)

Identification and validation of NFIA as a novel prognostic marker in renal cell carcinoma.

Prognostic tools are an essential component of the clinical management of patients with renal cell carcinoma (RCC). Although tumour stage and grade can provide important information, they fail to consider patient- and tumour-specific biology. In this study, we set out to find a novel molecular marker of RCC by using hepatocyte nuclear factor 4A (HNF4A), a transcription factor implicated in RCC progression and malignancy, as a blueprint. Through transcriptomic analyses, we show that the nuclear factor I A (NFIA)-driven transcription network is active in primary RCC and that higher levels of NFIA confer a survival benefit. We validate our findings using immunohistochemical staining and analysis of a 363-patient tissue microarray (TMA), showing for the first time that NFIA can independently predict poor cancer-specific survival in clear cell RCC (ccRCC) patients (hazard ratio=0.46, 95% CI=0.24-0.85, p value=0.014). Furthermore, we confirm the association of HNF4A with higher grades and stages in ccRCC in our TMA cohort. We present novel data that show HNF4A protein expression does not confer favourable prognosis in papillary RCC, confirming our survival analysis with publicly available HNF4A RNA expression data. Further work is required to elucidate the functional role of NFIA in RCC as well as the testing of these markers on patient material from diverse multi-centre cohorts, to establish their value for the prognostication of RCC.

Phenotypic Spectrum of NFIA Haploinsufficiency: Two Additional Cases and Review of the Literature.

The NFIA (nuclear factor I/A) gene encodes for a transcription factor belonging to the nuclear factor I family and has key roles in various embryonic differentiation pathways. In humans, NFIA is the major contributor to the phenotypic traits of "Chromosome 1p32p31 deletion syndrome". We report on two new cases with deletions involving NFIA without any other pathogenic protein-coding gene alterations. A cohort of 24 patients with NFIA haploinsufficiency as the sole anomaly was selected by reviewing the literature and public databases in order to analyze all clinical features reported and their relative frequencies. This process was useful because it provided an overall picture of the phenotypic outcome of NFIA haploinsufficiency and helped to define a cluster of phenotypic traits that can facilitate clinicians in identifying affected patients. NFIA haploinsufficiency can be suspected by a careful observation of the dysmorphisms (macrocephaly, craniofacial, and first-finger anomalies), and this potential diagnosis is strengthened by the presence of intellectual and developmental disabilities or other neurodevelopmental disorders. Further clues of NFIA haploinsufficiency can be provided by instrumental tests such as MRI and kidney urinary tract ultrasound and confirmed by genetic testing.

MiR-181d-5p Protects against Retinal Ganglion Cell Death after Blunt Ocular Injury by Regulating NFIA-Medicated Astrocyte Development.

Background Traumatic optic neuropathy (TON) refers to damage to the optic nerve resulting from direct and indirect trauma to the head and face. One of the important pathological processes in TON is the death of retinal ganglion cells (RGCs), but the cause of RGCs death remains unclear. We aimed to explore the mechanisms of RGCs death in an experimental TON model. Methods Optic nerve crush injury was induced in ten New Zealand white rabbits. On the 1st, 3rd, 7th, 14th, and 28th days after the operation, the retinal tissues of the rabbits were observed pathologically by hematoxylin-eosin staining. The expression of POU-homeodomain transcription factor Brn3a and glial fibrillary acidic protein (GFAP) was measured by immunofluorescence to evaluate the number of RGCs and astrocytes, respectively. miRNA expression and protein levels were assessed by RT-qPCR and western blot methods, respectively. Finally, the malondialdehyde content, superoxide dismutase activity, and proinflammatory factor levels were measured by ELISA. Western blot and dual-luciferase reporter assays were used to elucidate the relationship between miR-181d-5p and nuclear factor I-A (NFIA). Results Blunt ocular trauma increased oxidative stress and apoptosis and reduced ganglion cell layer (GCL) density. The expression of miR-181d-5p was decreased in retinal tissues, and its overexpression relieved RGCs death, astrocyte development, oxidative stress, and inflammation of the retina, which were reversed by NFIA overexpression. Conclusion miR-181d-5p can protect against the deterioration of TON by inhibiting RGCs death, astrocyte development, oxidative stress, and inflammation by targeting NFIA. This study provides new insight into early medical intervention in patients with TON.

NFIA determines the cis-effect of genetic variation on Ucp1 expression in murine thermogenic adipocytes

SummaryThermogenic brown and beige adipocytes counteract obesity by enhancing energy dissipation via uncoupling protein-1 (Ucp1). However, the effect of genetic variation on these cells, a major source of disease susceptibility, has been less well studied. Here we examined beige adipocytes from obesity-prone C57BL/6J (B6) and obesity-resistant 129X1/SvJ (129) mouse strains and identified a cis-regulatory variant rs47238345 that is responsible for differential Ucp1 expression. The alternative T allele of rs47238345 at the Ucp1 -12kb enhancer in 129 facilitates the allele-specific binding of nuclear factor I-A (NFIA) to mediate allele-specific enhancer-promoter interaction and Ucp1 transcription. Furthermore, CRISPR-Cas9/Cpf1-mediated single nucleotide polymorphism (SNP) editing of rs47238345 resulted in increased Ucp1 expression. We also identified Lim homeobox protein 8 (Lhx8), whose expression is higher in 129 than in B6, as a trans-acting regulator of Ucp1 in mice and humans. These results demonstrate the cis- and trans-acting effects of genetic variation on Ucp1 expression that underlie phenotypic diversity.

Association of ARRDC3 and NFIA variants with bovine congestive heart failure in feedlot cattle

Background: Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease. Methods: A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar’s test. Results: The most significant genome-wide association was in the arrestin domain-containing protein 3 gene (ARRDC3), followed by the nuclear factor IA gene (NFIA, mid-p-values, 1x10-8 and 2x10-7, respectively). Animals with homozygous risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates without those risk alleles (CI95 = 3-17). Animals with homozygous risk alleles at both genes were 28-fold more likely to have BCHF than all others (p-value = 1x10-7, CI95 = 4-206). A linked missense variant in ARRDC3 (C182Y) represents a potential functional variant as the C182 codon is conserved among all other jawed vertebrate species observed. A DNA test with two markers showed 29% of 273 BCHF cases had homozygous risk alleles in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This DNA test may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here. Conclusions: Although pathogenic roles for ARRDC3 and NFIA variants associated with BCHF are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management.

An miRNA derived from amelogenin exon4 regulates expression of transcription factor Runx2 by directly targeting upstream activators Nfia and Prkch

Amel, the gene encoding the amelogenin protein involved in enamel formation, is highly alternatively spliced. When exon4 is excised, it can form a mature miRNA (miR-exon4) that has previously been suggested to indirectly regulate expression of the Runt-related transcription factor 2 (Runx2) involved in bone development in ameloblasts and osteoblasts. However, the precise mechanism of this regulation is unclear. In this study, we aimed to identify direct targets of miR-exon4. The transcription factor family nuclear factor I/A (NFI/A) is known to negatively regulate expression of Runx2 and is among the most highly predicted direct targets of miR-exon4 that link to Runx2. Immunostaining detected NFI/A in osteoblasts and ameloblasts in vivo, and reporter assays confirmed direct interaction of the Nfia 3′-UTR and miR-exon4. In addition, silencing of Nfia in MC3T3-E1-M14 osteoblasts resulted in subsequent downregulation of Runx2. In a monoclonal subclone (mi2) of MC3T3-E1 cells wherein mature miR-exon4 was functionally inhibited, we observed significantly downregulated Runx2 expression. We showed that NFI/A was significantly upregulated in mi2 cells at both mRNA and protein levels. Furthermore, quantitative proteomics and pathway analysis of gene expression in mi2 cells suggested that miR-exon4 could directly target Prkch (protein kinase C-eta), possibly leading to RUNX2 regulation through mechanistic target of rapamycin kinase activation. Reporter assays also confirmed the direct interaction of miR-exon4 and the 3′-UTR of Prkch, and Western blot analysis confirmed significantly upregulated mechanistic target of rapamycin kinase phosphorylation in mi2 cells. Taken together, we conclude that Nfia and Prkch expression negatively correlates with miR-exon4-mediated Runx2 regulation in vivo and in vitro, suggesting miR-exon4 directly targets Nfia and Prkch to regulate Runx2.

Nuclear Factor IA Is Down-regulated in Muscle-invasive and High-grade Bladder Cancers.

Nuclear factor I (NFI) A and NFIB are transcription factors involved in the regulation of cell differentiation and organ development. More recently, they have been implicated in the pathogenesis of cancer, acting as context-dependent tumor promoters or suppressors. Expression of NFIA and NFIB was assessed by immunohistochemistry in 136 primary urothelial bladder cancers. Progressive down-regulation of NFIA was observed with increasing pT stages and higher grade of analyzed tumors. Consequently, muscle invasive cancers exhibited lower NFIA expression compared with non-muscle invasive cases. Analogous comparisons yielded negative results in the case of NFIB. Expression of neither protein was associated with patient survival. NFIA may act as a suppressor of urothelial carcinogenesis, but functional studies and understanding of post-transcriptional regulation of NFI expression is necessary to dissect its role in bladder malignancies.

Expression and role of microRNA-212/nuclear factor I-A in depressive mice

ABSTRACT Depression is characterized by persistent depressed mood and cognitive dysfunction, severely impacting human health. In the present study, we aimed to explore the role and mechanism of microRNA (miR)-212 in depression in vivo. Chronic unpredictable mild stress (CUMS) mice were established, and depression-like behaviors were confirmed using the forced swimming test (FST), sucrose preference test (SPT), and the tail suspension test (TST). Next, the expression of miR-212 and its potential target, i.e., nuclear factor I-A (NFIA), was verified using quantitative reverse transcription (qRT)-PCR analysis and Western blotting in CUMS mice. The effects of miR-212 and NFIA on depression-like behaviors, inflammatory response, and neuronal apoptosis were examined using FST, TST, SPT, enzyme-linked immunosorbent assay (ELISA) assay, and flow cytometry analysis. Finally, the relationship between miR-212 and NFIA was examined using a dual-luciferase reporter assay. Based on our findings, miR-212 was significantly upregulated, while NFIA was downregulated in CUMS mice. miR-212 overexpression could suppress the CUMS-induced weight loss, immobility time in FST and TST, and increased hippocampal neuronal apoptosis and pro-inflammatory cytokines levels. In addition, NFIA upregulation could partially reverse the effects of miR-212 mimic in CUMS mice. Accordingly, miR-212 could ameliorate CUMS-induced depression-like behavior in mice by targeting NFIA, indicating its protective role in depression.

MicroRNA-361-5p Aggravates Acute Pancreatitis by Promoting Interleukin-17A Secretion via Impairment of Nuclear Factor IA-Dependent Hes1 Downregulation.

This study set out to explore the potential role of microRNA-361-5p (miR-361-5p) in acute pancreatitis through regulation of interleukin-17A (IL-17A). We first identified the expression of miR-361-5p, IL-17A, nuclear factor IA (NFIA), and hes family bHLH transcription factor 1 (Hes1) in serum samples collected from patients with acute pancreatitis, caerulein-induced mice, and a Th17 cell model. The predicted binding of miR-361-5p to NFIA was confirmed in vitro. Gain- and loss-of-function assays of miR-361-5p and NFIA were employed to elucidate their effects on acute pancreatitis. miR-361-5p promoted Th17 cells to secrete IL-17A and then aggravated acute pancreatitis. miR-361-5p directly targeted NFIA by binding to its promoter region, leading to its downregulation. Overexpression of NFIA reduced Hes1 expression and rescued the promoting effect of miR-361-5p on IL-17A secretion. In summary, miR-361-5p enhances IL-17A secretion from Th17 cells and thus aggravates acute pancreatitis by targeting NFIA and upregulating Hes1.

SoxE group transcription factor Sox8 promotes astrocytic differentiation of neural stem/precursor cells downstream of Nfia.

The brain consists of three major cell types: neurons and two glial cell types (astrocytes and oligodendrocytes). Although they are generated from common multipotent neural stem/precursor cells (NS/PCs), embryonic NS/PCs cannot generate all of the cell types at the beginning of brain development. NS/PCs first undergo extensive self‐renewal to expand their pools, and then acquire the potential to produce neurons, followed by glial cells. Astrocytes are the most frequently found cell type in the central nervous system (CNS), and play important roles in brain development and functions. Although it has been shown that nuclear factor IA (Nfia) is a pivotal transcription factor for conferring gliogenic potential on neurogenic NS/PCs by sequestering DNA methyltransferase 1 (Dnmt1) from astrocyte‐specific genes, direct targets of Nfia that participate in astrocytic differentiation have yet to be completely identified. Here we show that SRY‐box transcription factor 8 (Sox8) is a direct target gene of Nfia at the initiation of the gliogenic phase. We found that expression of Sox8 augmented leukemia inhibitory factor (LIF)‐induced astrocytic differentiation, while Sox8 knockdown inhibited Nfia‐enhanced astrocytic differentiation of NS/PCs. In contrast to Nfia, Sox8 did not induce DNA demethylation of an astrocyte‐specific marker gene, glial fibrillary acidic protein (Gfap), but instead associated with LIF downstream transcription factor STAT3 through transcriptional coactivator p300, explaining how Sox8 expression further facilitated LIF‐induced Gfap expression. Taken together, these results suggest that Sox8 is a crucial Nfia downstream transcription factor for the astrocytic differentiation of NS/PCs in the developing brain.

The miR-223/nuclear factor I-A axis regulates inflammation and cellular functions in intestinal tissues with necrotizing enterocolitis.

We previously demonstrated that microRNA(miR)‐223 is overexpressed in intestinal tissue of infants with necrotizing enterocolitis (NEC). The objective of the current study was to identify the target gene of miR‐223 and to investigate the role of the miR‐223/nuclear factor I‐A (NFIA) axis in cellular functions that underpin the pathophysiology of NEC. The target gene of miR‐223 was identified by in silico target prediction bioinformatics, luciferase assay, and western blotting. We investigated downstream signals of miR‐223 and cellular functions by overexpressing the miRNA in Caco‐2 and FHs74 cells stimulated with lipopolysaccharide or lipoteichoic acid (LTA). NFIA was identified as a target gene of miR‐223. Overexpression of miR‐223 significantly induced MYOM1 and inhibited NFIA and RGN in Caco‐2 cells, while costimulation with LTA decreased expression of GNA11, MYLK, and PRKCZ. Expression levels of GNA11, MYLK, IL‐6, and IL‐8 were increased, and levels of NFIA and RGN were decreased in FHs74 cells. These potential downstream genes were significantly correlated with levels of miR‐223 or NFIA in primary NEC tissues. Overexpression of miR‐223 significantly increased apoptosis of Caco‐2 and FHs74 cells, while proliferation of FHs74 was inhibited. These results suggest that upon binding with NFIA, miR‐223 regulates functional effectors in pathways of apoptosis, cell proliferation, G protein signaling, inflammation, and smooth muscle contraction. The miR‐223/NFIA axis may play an important role in the pathophysiology of NEC by enhancing inflammation and tissue damage.

Recurrent NFIA K125E substitution represents a loss-of-function allele: Sensitive in vitro and in vivo assays fornontruncating alleles.

Nuclear factor I A (NFIA) is a transcription factor that belongs to the NFI family. Truncating variants or intragenic deletion of the NFIA gene are known to cause the human neurodevelopmental disorder known as NFIA‐related disorder, but no patient heterozygous for a missense mutation has been reported. Here, we document two unrelated patients with typical phenotypic features of the NFIA‐related disorder who shared a missense variant p.Lys125Glu (K125E) in the NFIA gene. Patient 1 was a 6‐year‐old female with global developmental delay, corpus callosum anomaly, macrocephaly, and dysmorphic facial features. Patient 2 was a 14‐month‐old male with corpus callosum anomaly and macrocephaly. By using Drosophila and zebrafish models, we functionally evaluated the effect of the K125E substitution. Ectopic expression of wild‐type human NFIA in Drosophila caused developmental defects such as eye malformation and premature death, while that of human NFIA K125E variant allele did not. nfia‐deficient zebrafish embryos showed defects of midline‐crossing axons in the midbrain/hindbrain boundary. This impairment of commissural neurons was rescued by expression of wild‐type human NFIA, but not by that of mutant variant harboring K125E substitution. In accordance with these in vivo functional analyses, we showed that the K125E mutation impaired the transcriptional regulation of HES1 promoter in cultured cells. Taken together, we concluded that the K125E variant in the NFIA gene is a loss‐of‐function mutation.

MicroRNA miR-212-3p targets NFIA to suppress HBV replication and tumor progression in hepatocellular carcinoma via repressing Enhancer I activity

IntroductionEmerging evidence identifies that microRNAs (miRNAs) are associated with hepatitis B virus (HBV) infection. In the current study, we mainly focus on the functions and underlying mechanisms of miR-212-3p in HBV replication in hepatocellular carcinoma (HCC).Material and methodsThe levels of miR-212-3p, nuclear factor I A (NFIA) and HBV DNA copies were measured by qRT-PCR. The level of core particle-associated HBV DNA, the productions of hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg), and the expression of NFIA were detected via southern blot assay, ELISA and western blot assay, respectively. The putative target of miR-212-3p was predicted by TargetScan and Pictar, followed by the dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay to validate the interaction. The interaction between miR-212-3p and Enhancer I/X promoter (EnI/Xp) reporter was also verified by dual luciferase reporter assay. In addition, the cell viability and apoptotic rate were detected by MTT and flow cytometry, respectively.ResultsmiR-212-3p mimics or NFIA knockdown inhibited HBV expression and replication in HepG2.2.15 cells, while miR-212-3p inhibitor or NFIA overexpression showed the opposite trend. NFIA was confirmed as a direct target of miR-212-3p. Furthermore, miR-212-3p impeded HBV expression and replication by suppressing NFIA. Besides, miR-212-3p lowered EnI/Xp activity by regulating NFIA. In addition, miR-212-3p retarded cell viability and induced apoptosis through targeting NFIA.ConclusionsmiR-212-3p targets NFIA to down-regulate its expression, thereby inhibiting HBV replication and tumorigenesis in HCC. Our finding might provide a promising therapeutic target for HBV infection.