Interferon-induced Protein With Tetratricopeptide Repeats 2 Research Articles

Genetic polymorphisms in innate immunity genes influence predisposition to tick-borne encephalitis

Tick-borne encephalitis (TBE) is a neuroviral disease that ranges in severity from a mild febrile illness to a severe and life-threatening meningoencephalitis or encephalomyelitis. There is increasing evidence that susceptibility to tick-borne encephalitis virus (TBEV)-induced disease and its severity are largely influenced by host genetic factors, in addition to other virus- and host-related factors. In this study, we investigated the contribution of selected single nucleotide polymorphisms (SNPs) in innate immunity genes to predisposition to TBE in humans. More specifically, we investigated a possible association between SNPs rs304478 and rs303212 in the gene Interferon Induced Protein With Tetratricopeptide Repeats 1 (IFIT1), rs7070001 and rs4934470 in the gene Interferon Induced Protein With Tetratricopeptide Repeats 2 (IFIT2), and RIG-I (Retinoic acid-inducible gene I) encoding gene DDX58 rs311795343, rs10813831, rs17217280 and rs3739674 SNPs with predisposition to TBE in population of the Czech Republic, where TBEV is highly endemic. Genotypic and allelic frequencies for these SNPs were analyzed in 247 nonimmunized TBE patients and compared with 204 control subjects. The analysis showed an association of IFIT1 rs304478 SNP and DDX58 rs3739674 and rs17217280 SNPs with predisposition to TBE in the Czech population indicating novel risk factors for clinical TBE but not for disease severity. These results also highlight the role of innate immunity genes in TBE pathogenesis.

MiR-199a-5P promotes osteogenic differentiation of human stem cells from apical papilla via targeting IFIT2 in apical periodontitis.

Periapical alveolar bone loss is the common consequence of apical periodontitis (AP) caused by persistent local inflammation around the apical area. Human stem cells from apical papilla (hSCAPs) play a crucial role in the restoration of bone lesions during AP. Studies have recently identified the critical role of microRNAs (miRNAs) involved in AP pathogenesis, but little is known about their function and potential molecular mechanism, especially in the osteogenesis of hSCAPs during AP. Here, we investigated the role of clinical sample-based specific miRNAs in the osteogenesis of hSCAPs. Differential expression of miRNAs were detected in the periapical tissues of normal and patients with AP via transcriptomic analysis, and the expression of miR-199a-5p was confirmed by qRT-PCR. Treatment of hSCAPs with miR-199a-5p mimics while loaded onto beta-tricalcium phosphate (β-TCP) ceramic particle scaffold to explore its effect on osteogenesis in vivo. RNA binding protein immunoprecipitation (RIP) and Luciferase reporter assay were conducted to identify the target gene of miR-199a-5p. The expression of miR-199a-5p was decreased in the periapical tissues of AP patients, and miR-199a-5p mimics markedly enhanced cell proliferation and osteogenic differentiation of hSCAPs, while miR-199a-5p antagomir dramatically attenuated hSCAPs osteogenesis. Moreover, we identified and confirmed Interferon Induced Protein with Tetratricopeptide Repeats 2 (IFIT2) as a specific target of miR-199a-5p, and silencing endogenous IFIT2 expression alleviated the inhibitory effect of miR-199a-5p antagomir on the osteogenic differentiation of hSCAPs. Furthermore, miR-199a-5p mimics transfected hSCAPs loaded onto beta-tricalcium phosphate (β-TCP) scaffolds induced robust subcutaneous ectopic bone formation in vivo. These results strengthen our understanding of predictors and facilitators of the key AP miRNAs (miR-199a-5p) in bone lesion repair under periapical inflammatory conditions. And the regulatory networks will be instrumental in exploring the underlying mechanisms of AP and lay the foundation for future regenerative medicine based on dental mesenchymal stem cells.

The Involvement of Insulin-Like Growth Factor 2 Messenger Ribonucleic Acid-Binding Protein 2 in the Regulation of the Expression of Breast Cancer-Related Genes.

AimThis study investigated the role and mechanism of insulin-like growth factor 2-IGF2BP2 in breast cancer.MethodsIGF2BP2 is overexpressed in MDA-MB-231 human breast cancer cells. Thus, RNA sequencing was used to analyze the differentially expressed genes, Cell Counting Kit-8 was used to detect cell proliferation, and a Transwell assay was used to assess cell invasion. Following on from the RNA sequencing results, Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), chemokine C-C motif ligand 20 (CCL20), chemokine C-C motif ligand 5 (CCL5), and chemokine C-X-C motif ligand 10 (CXCL10) regulated by IGF2BP2 were subjected to real-time reverse transcriptase-polymerase chain reaction verification.ResultsAfter IGF2BP2 overexpression, 67 genes were up-regulated, and 87 genes were down-regulated. The gene with the most significant up-regulation was homeobox protein 1 (PROX1), and the gene with the most significant down-regulation was Acidic β-crystallin 4 (CRYBA4). The most enriched gene ontology (GO) terms of up-regulated differentially expressed genes are protein binding and cell membrane and of down-regulated differentially expressed genes they are ion binding, cytoplasm, and response to virus. Kyoto Encyclopedia of Genes and Genomes analysis showed that the up-regulated differential genes were mainly enriched in protein processing, the endoplasmic reticulum, and the regulation of actin cytoskeleton, while down-regulated differential genes were mainly enriched in rheumatoid arthritis, chemokine signaling pathways, toll-like receptor signaling pathways, tumor necrosis factor signaling pathways, cytokine-cytokine receptor interaction, and Notch signaling pathways. IGF2BP2 overexpression significantly promoted the proliferation and invasion of breast cancer cells (P < 0.01). Compared with the control group, the IGF2BP2 overexpression group had significantly increased expressions of IFIT2, CCL20, and CXCL10 (P < 0.05).ConclusionIGF2BP2 may promote the invasion and proliferation of human breast cancer cells by up-regulating breast cancer-related genes, such as IFIT2, CCL20, and CXCL10.

SAHA Overcomes 5-FU Resistance in IFIT2-Depleted Oral Squamous Cell Carcinoma Cells.

Simple SummaryIFIT2 depletion is associated with increased epithelial-mesenchymal transition and metastasis. The main aim of our study was to understand the link between drug resistance and IFIT2 depletion. In this study, we confirmed resistance to multiple common therapeutic drugs, particularly 5-FU, which showed especially high resistance in IFIT2-depleted cells. Interestingly, combination of SAHA and 5-FU overcame 5-FU resistance in IFIT2-depleted cells. Hence, our findings suggest that IFIT2 expression may be used as a biomarker to decide whether to undergo 5-FU treatment, but also the SAHA and 5-FU combination may be a potential new treatment regimen to augment 5-FU therapy in patients with thymidylate synthase-mediated drug-resistant oral squamous cell carcinoma.Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) is a member of the interferon-stimulated gene family that contains tetratricopeptide repeats (TPRs), which mediate protein–protein interactions in various biological systems. We previously showed the depletion of IFIT2 enhanced cell migration and metastatic activity in oral squamous cell carcinoma (OSCC) cells via the activation of atypical PKC signaling. In this study, we found that IFIT2-knockdown cells displayed higher resistance to 5-fluorouracil (5-FU) than control cells. The comet assay and annexin V analysis showed decreased DNA damage and cell death in IFIT2-knockdown cells compared to control cells treated with 5-FU. Cell cycle progression was also perturbed by 5-FU treatment, with the accumulation of IFIT2-depleted cells in S phase in a time-dependent manner. We further observed the overexpression of thymidylate synthase (TS) and thymidine kinase (TK) in IFIT2-knockdown cells. Inhibition of TS alone or double inhibition of TS and TK1 using the siRNA technique increased susceptibility to 5-FU in IFIT2-knockdown cells. We further identified that suberanilohydroxamic acid (SAHA) treatment decreased the expression of TS in IFIT2-knockdown cells and demonstrated that pretreatment with SAHA sensitized IFIT2-knockdown cells to 5-FU in vitro and in vivo. In conclusion, IFIT2 knockdown enhances TS expression, which mediates 5-FU resistance, and SAHA pretreatment suppresses TS expression and hence sensitizes cells to 5-FU. SAHA will be an effective strategy for the treatment of OSCC patients with 5-FU resistance.

The expression of interferon-induced protein with tetratricopeptide repeats 2 in gastric cancer tissue and prognostic significance

Objective To discuss the expression of interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) in gastric cancer tissue, and to analyze the correlation of expression level of IFIT2 and clinical features and prognosis of gastric cancer patients. Methods Immunohistochemical staining method was used to detect the expression of IFIT2 in 90 cases of gastric cancer tissue and corresponding normal adjacent tissues in tissue chip. The rank sum test was used to compare the difference in the expression level of IFIT2 in gastric cancer tissue and corresponding normal adjacent tissue, and chi-square test was used to analyze the correlation of expression level of IFIT2 and clinical features. Kaplan-Meier method was used to analyze the correlation of expression level of IFIT2 and overall survival, and Cox model was fitted to estimate the correlation of different indexes and prognosis. Results The high expression rate of IFIT2 in gastric cancer tissue (77.4%) was significantly higher than that in normal adjacent tissue (95.20%, χ2=11.334, P=0.001). The high expression rate of IFIT2 in stage Ⅲ-Ⅳ (72.00%, 36/50) was significantly lower than that in stage Ⅰ-Ⅱ (90.91%, 30/33; χ2=4.364, P=0.037). Patients with higher expression level of IFIT2 had longer OS than that with lower expression level with the difference being statistically significant (χ2=10.990, P=0.001). The Cox regression model indicated that distant metastasis [hazard ratio(HR)=2.693, 95% confidence interval (CI): 1.164-6.232, P=0.021], and IFIT2 high expression (HR=0.350, 95%CI: 0.169-0.727, P=0.005) were independent prognostic factors of gastric cancer. Conclusion IFIT2 had important effect in initiation and development of gastric cancer, and could serve as an important risk factor to predcit gastric cancer prognosis. Key words: Gastric cancer; Interferon-induced protein with tetratricopeptide repeats 2; Prognosis

Curcumin induces apoptosis in human leukemic cell lines through an IFIT2-dependent pathway

ABSTRACTCurcumin, the primary bioactive component isolated from turmeric, has been shown to possess variety of biologic functions including anti-cancer activity. However, molecular mechanisms in different cancer cells are various. In the present study, we demonstrated that curcumin induced G2/M cell cycle arrest and apoptosis by increasing the expression levels of cleaved caspase-3, cleaved PARP and decreasing the expression of BCL−2 in U937 human leukemic cells but not in K562 cells. We found some interferon induced genes, especially interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), were significantly upregulated when treated with curcumin in U937 cells by gene expression chip array, and further confirmed that the expression of IFIT2 was obviously higher in U937 than that in K562 cells by Western blot assay. In addition, inhibiting the expression of IFIT2 by shRNA in U937 rescued curcumin-induced apoptosis and exogenous overexpression of IFIT2 by lentiviral transduction or treating with IFNγ in K562 cells enhanced anti-cancer activity of curcumin. These results indicated for the first time that curcumin induced leukemic cell apoptosis via an IFIT2-dependent signaling pathways. The present study identified a novel mechanism underlying the antitumor effects of curcumin, and may provide a theoretical basis for curcumin combined with interferon in the cancer therapeutics.

Blocking TNF-α inhibits angiogenesis and growth of IFIT2-depleted metastatic oral squamous cell carcinoma cells

Our previous study demonstrated that the depletion of interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) promoted metastasis and was associated with a poor prognosis in patients with oral squamous cell carcinoma (OSCC). Our current study explores the major downstream signaling involved in IFIT2 depletion-induced OSCC metastasis. To this end, we used two cell lines (designated sh-control-xeno and sh-IFIT2-xeno) derived from human OSCC xenografts expressing sh-control and sh-IFIT2, respectively, and one metastatic OSCC subline (sh-IFIT2-meta) from an IFIT2-depleted metastatic tumor. We found that the sh-IFIT2-meta cells proliferated more slowly than the sh-control-xeno cells but exhibited higher migration and chemoresistance. Using microarray technology and Ingenuity Pathway Analysis, we found that TNF-α was one of the major downstream targets in IFIT2-depleted OSCC cells. Quantitative real-time PCR, western blotting, and ELISA results confirmed that TNF-α was upregulated in the sh-IFIT2-meta cells. Blocking TNF-α abolished the angiogenic activity induced by the sh-IFIT2-meta cells. Furthermore, the human-specific TNF-α antibody golimumab significantly inhibited in vivo angiogenesis, tumor growth and metastasis of sh-IFIT2-meta cells. These results demonstrate that IFIT2 depletion results in TNF-α upregulation, leading to angiogenesis and metastasis of OSCC cells.

MicroRNA-645, up-regulated in human adencarcinoma of gastric esophageal junction, inhibits apoptosis by targeting tumor suppressor IFIT2.

BackgroundAn increasing body of evidence indicates that miRNAs have a critical role in carcinogenesis and cancer progression; however, the role of miRNAs in the tumorigenesis of adencarcinoma of gastric esophageal junction (AGEJ) remains largely unclear.MethodsThe SGC7901 and BGC-823 gastric cancer cell lines were used. The expressions of miR-645 and IFIT2 (Interferon-induced protein with tetratricopeptide repeats 2) were examined by qRT-PCR, The expressions of IFIT2 was examined by western blotting and immunohistochemistry assay. The cell apoptosis was determined by FACS. MiR-645 inhibitor, mimics and plasmid-IFIT2 transfections were performed to study the loss- and gain-function. Caspase-3/7 activity was examined by caspase-3/7 assay.ResultsIn the present study, we have reported an increased expression of miR-645 in AGEJ clinical specimens compared with paired non-cancerous tissues. We also observed a significant miR-645 up-regulation in two gastric cancer (GC) cell lines, SGC7901 and BGC-823, which were used as cell models because there was no available AGEJ cell lines established to date. We found that inhibition of miR-645 could sensitize dramatically SGC7901 and BGC-823 cells to both serum starvation– and chemotherapeutic drug–induced apoptosis by up-regulating IFIT2, a mediator of apoptosis via a mitochondrial pathway, with a potential binding site for miR-645 in its mRNA’s 3′UTR. Further investigation exhibited that IFIT2 expression decreases in SGC7901 and BGC-823 cells and AGEJ tissues. IFIT2 ectopic expression leads to promotion of cell apoptosis, indicating that IFIT2 may function as a suppressor in the development of AGEJ. Furthermore, inhibition of miR-645 induces up-regulation of IFIT2 and increased caspase-3/7 activity compared with control groups.ConclusionsOur data suggest that miR-645 functions as an oncogene in human AGEJ by, at least partially through, targeting IFIT2.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-633) contains supplementary material, which is available to authorized users.

New Insight into Human Disc Degeneration by Gene Expression Profiling

Introduction Intervertebral disc (IVD) degeneration is characterized by the breakdown of extracellular matrix molecules and is often associated with inflammatory processes. Hence, anabolic, anti-catabolic, or anti-inflammatory treatments have been considered to retard or reverse early degenerative changes in the IVD. However, there is still a lack of fundamental knowledge about the molecular transformations in the degenerative compared with the native healthy discs. More detailed understanding of the molecular mechanisms will be essential to develop specific therapeutic strategies. In the present study, microarray and quantitative gene expression analysis were used to compare expression profiles of cells from healthy and degenerative human IVDs. The aim is to identify significantly dysregulated molecules or pathways that can potentially be targeted for regenerative therapy. Materials and Methods Annulus fibrosus (AF) and nucleus pulposus (NP) tissues were obtained from human lumbar discs through organ donation program and in accordance with the local and institutional ethical guidelines. Harvested tissue was assigned to either the “healthy” (Thompson disc degeneration grade I-II) or the “degenerative” group (Thompson grade III-IV). Cells were isolated from tissues using sequential pronase and collagenase digestion. Total RNA was extracted from isolated cells using a TRI-Spin method. Samples were processed and subjected to Affymetrix Whole Human Genome DNA microarray profiling. After correspondence analysis for data correction, expression differences between healthy and degenerative AF and NP cells, respectively, were analyzed. Genes with most significant expression divergences were further assessed using quantitative real time RT-PCR. Results For both NP and AF, n = 8 healthy and n = 16 degenerative RNA samples were profiled by microarray. Biostatistical analysis revealed that 237 genes were differentially regulated between degenerative and healthy human AF cells, while 178 genes were differentially regulated between degenerative and healthy NP cells. Specifically, 119 (AF) and 66 (NP) genes were found up-regulated, whereas 118 (AF) and 112 (NP) genes were down-regulated in the degenerative group. Quantitative gene expression analysis was performed for the most significantly differentially regulated genes in n = 8 healthy and n = 10 degenerative RNA samples. While some genes were specifically modulated in the AF or NP cells, several genes showed significant differential regulation ( p &lt; 0.05) by degeneration status. Genes up-regulated in degenerative compared with healthy IVD cells included activated leukocyte cell adhesion molecule (ALCAM), cyclin D1 (CCND1), insulin-like growth factor binding protein 3 (IGFBP3), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), magnesium transporter 1 (MAGT1), and tissue factor pathway inhibitor (TFPI). Significantly down-regulated genes in degenerative compared with healthy IVD cells, include dickkopf 1 homolog (DKK1), forkhead box F2 (FOXF2), lectin galactoside-binding-like (LGALSL), lipoprotein lipase (LPL), and mannosidase (MAN2B2). Moreover, modulation of distinct molecular pathways could be recognized. Conclusion The present data demonstrates upregulation of signaling factors and antagonists, growth factors and their inhibitors, and chemotactic factors in degenerative cells. Moreover, factors involved in matrix turnover were down-regulated. Improved insight in the differential regulation of distinct molecules and pathways may ultimately allow specific treatment on a molecular basis. Further validation at the protein expression level will be required to evaluate their potential as therapeutic target or as biomarkers for predicting the state of degenerative process in the discs. Disclosure of Interest S. Grad: Conflict with AO Foundation Collaborative Research Program Annulus Fibrosus Repair R. Gawri: None declared L. Haglund: None declared J. Ouellet: Conflict with DePuySynthes, AO Foundation, AONA F. Mwale: None declared L. Creemers: Conflict with Dutch Arthritis Association J. Rutges: None declared W. Gallagher: None declared P. O'Gaora: None declared Pandit: None declared M. Alini: Conflict with AO Foundation Collaborative Research Program Annulus Fibrosus Repair

Depleting IFIT2 mediates atypical PKC signaling to enhance the migration and metastatic activity of oral squamous cell carcinoma cells

Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) is one of the most highly responsive interferon-stimulated genes, but its biological functions are poorly understood. In this study, we aimed to explore the underlying mechanisms by which depleting IFIT2 induces the migration of oral squamous cell carcinoma (OSCC) cells. Stable IFIT2-depleted cells underwent epithelial-mesenchymal transition (EMT) and exhibited enhanced cell motility and invasiveness compared with control cells. Furthermore, our results indicated that atypical protein kinase C (aPKC) was activated in IFIT2-depleted cells. Inhibition of aPKC using a specific myristoylated PKCζ pseudosubstrate or aPKC-targeting small interfering RNA (siRNA) abolished IFIT2 depletion-induced EMT, migration and invasion, indicating that the activation of aPKC has an essential role in regulating the cellular responses induced by IFIT2 depletion. Following tail-vein injection, IFIT2-depleted OSCC cells colonized not only the lungs but also the heart, head and neck, retroperitoneal, and peritoneal cavities; whereas control cells predominantly localized in the lungs. IFIT2 mRNA and protein expression was positively associated with E-cadherin expression in OSCC patient specimens. The loss of E-cadherin and IFIT2 expression was observed at the invasive front of OSCC tumors, suggesting that the loss of IFIT2 may induce EMT and lead to the metastasis of OSCCs. OSCC patients possessing reduced IFIT2-expression levels (IFIT2 <50%) exhibited greater rates of distant metastasis and poor prognoses compared with OSCC patients who expressed greater levels of IFIT2 (IFIT2 ≥50%). These results demonstrate that IFIT2 depletion activates the aPKC pathway and consequently induces EMT, cell migration and invasion. Most importantly, depleting IFIT2 may participate in OSCC tumor progression, particularly during metastasis. Taken together, our study demonstrates that IFIT2, a protein responsible for interferon stimulation, may prevent OSCC metastasis and serve as a valuable prognostic marker.

Seroreactivity against PTEN-induced putative kinase 1 (PINK1) in Turkish patients with Behçet's disease.

Behçet's disease (BD) is a multisystem inflammatory disorder characterized by recurrent oral ulcers, genital ulcers and ocular inflammation, as well as skin, joint, vascular, pulmonary, central nervous system (CNS) and gastrointestinal tract manifestations. The etiopathogenesis of BD has not yet been identified; but it has generally been accepted that several environmental factors may induce an inflammatory attack in genetically susceptible individuals. In this study, we aimed to identify antigens that could elicit high-titer IgG responses by the serological analysis of recombinant expression of cDNA libraries method (SEREX). We screened a human testis cDNA library with pooled sera obtained from 4 BD patients by SEREX. Antigens that were identified with the initial analysis were selected for seroreactivity analysis of a larger group of BD patients (n=78) and controls (n=66) by serological immunoscreening. We observed seroreactivity against 6 antigens using the pooled sera. These included rabaptin 5 (RABPT5), PTEN-induced putative kinase 1 (PINK1), switch associated protein 70 (SWAP70), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), ankyrin repeat domain 20 family, member A1 (ANKRD20A1), and an unknown antigen. Eleven out of 82 (13.4%) BD patients were found to have antibodies elicited against PINK1 antigen, when none of the control sera showed reactivity (p=0.001). There was no significant difference in the frequency of other defined antigens between the patient and control groups. However, among BD clinical sub-groups, anti-SWAP70 antibodies were found to associate with vascular involvement. In this study, antibodies against PINK1 were found to specifically associate with BD while SWAP70 antibody was associated with clinical sub-groups of BD. Although variations in both genetic background and environmental factors may affect the outcome of serological responses, our results suggest that serological screening can be used to identify antigens that elicit antibody responses associated with BD.

Analysis of gene-expression profiles by oligonucleotide microarray in children with influenza

In order to clarify the mechanism of the host response to influenza virus, gene-expression profiles of peripheral blood obtained from paediatric patients with influenza were investigated by oligonucleotide microarray. In the acute phase of influenza, 200 genes were upregulated and 20 genes were downregulated compared with their expression in the convalescent phase. Interferon-regulated genes, such as interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) and vipirin, were strongly upregulated in the acute phase. Gene ontology analysis showed that immune response genes were highly overrepresented among the upregulated genes. Gene-expression profiles of influenza patients with and without febrile convulsion were also studied. In patients with febrile convulsion, 22 genes were upregulated and five were downregulated compared with their expression in patients without febrile convulsion. These results should help to clarify the pathogenesis of influenza and its neurological complications.