Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity.

The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self versus non-self nucleic acid discrimination.

Physiological functions of RIG-I-like receptors.

RIG-I-like receptors (RLRs) are crucial for pathogen detection and triggering immune responses and have immense physiological importance. In this review, we first summarize the interferon system and innate immunity, which constitute primary and secondary responses. Next, the molecular structure of RLRs and the mechanism of sensing non-self RNA are described. Usually, self RNA is refractory to the RLR; however, there are underlying host mechanisms that prevent immune reactions. Studies have revealed that the regulatory mechanisms of RLRs involve covalent molecular modifications, association with regulatory factors, and subcellular localization. Viruses have evolved to acquire antagonistic RLR functions to escape the host immune reactions. Finally, the pathologies caused by the malfunction of RLR signaling are described.

Double-Stranded RNA Induces Mortality in an MDA5-Mediated Type I Interferonopathy Model.

Gain-of-function mutations in the viral dsRNA sensor melanoma differentiation-associated protein 5 (MDA5) lead to autoimmune IFNopathies, including Singleton-Merten syndrome (SMS) and Aicardi-Goutières syndrome. However, much remains unclear regarding the mechanism of disease progression and how external factors such as infection or immune stimulation with vaccination can affect the immune response. With this aim, we generated mice with human MDA5 bearing the SMS-associated mutation R822Q (hM-R822Q). hM-R822Q transgenic (Tg) mice developed SMS-like heart fibrosis, aortic valve enlargement, and aortic calcification with a systemic IFN-stimulated gene signature resulting in the activation of the adaptive immune response. Although administration of the viral dsRNA mimic polyinosinic-polycytidylic acid [poly(I:C)] did not have remarkable effects on the cardiac phenotype, dramatic inflammation was observed in the intestines where IFN production was most elevated. Poly(I:C)-injected hM-R822Q Tg mice also developed lethal hypercytokinemia marked by massive IL-6 levels in the serum. Interrupting the IFN signaling through mitochondrial antiviral signaling protein or IFN-α/ß receptor alleviated hM-R822Q-induced inflammation. Furthermore, inhibition of JAK signaling with tofacitinib reduced cytokine production and ameliorated mucosal damage, enabling the survival of poly(I:C)-injected hM-R822Q Tg mice. These findings demonstrate that the MDA5 R822Q mutant introduces a critical risk factor for uncontrollable inflammation on viral infection or vaccination.

Consistency Analysis of the UF-1500 and UF-5000 Automated Urine Particle Analyzers.

BACKGROUND: The aim was to evaluate the consistency of the results between the UF-1500 and UF-5000, fully automated urine particle analyzers. METHODS: A total of 554 randomly selected inpatient and outpatient urine samples were collected for analysis using the UF-1500, the UF-5000, and by manual microscopic examination. The coincidence rate, intraday repeatability, and interday reproducibility were evaluated on the UF-1500 and UF-5000. To analyze the review flags from the UF-1500, the UF-1500 results were compared to manual microscopy as the gold standard. RESULTS: The repeatability of red blood cells (RBCs), white blood cells (WBCs), epithelial cells (ECs), casts, and bacteria using the UF-1500 and UF-5000 is expressed as the relative standard deviations of the intraday and inter-day measurements. For the UF-1500, the relative standard deviation values ranged from 5.9% to 12.6% and 4.9% to 17.2% for the low and 1.6% to 9.3% and 2.3% to 16.9% for the high samples, respectively. The correlation co-efficient for RBCs, WBCs, ECs, SECs, casts, crystals, and bacteria for the UF-1500 were 0.981, 0.993, 0.968, 0.963, 0.821, 0.783, and 0.992, respectively. Review samples from the UF-1500 were confirmed by microscopic examination. Review flags for all 554 samples included 3 samples with "DEBRIS High" and 23 samples with "RBCs/YLC Abnormal classification". CONCLUSIONS: The identification of various urine components by both instruments meets laboratory requirements. These two instruments with different performances have specific characteristics and should be used based upon the needs of each laboratory.

Hepatitis B Virus (HBV) Upregulates TRAIL-R3 Expression in Hepatocytes Resulting in Escape From Both Cell Apoptosis and Suppression of HBV Replication by TRAIL.

BACKGROUND: Hepatitis B virus (HBV) evades host immunity by regulating intracellular signals. To clarify this immune tolerance mechanism, we performed gene expression analysis using HBV-infected humanized mouse livers. METHODS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 3 (TRAIL-R3) was significantly upregulated in livers of HBV-infected human hepatocyte transplanted mice by cDNA microarray and next-generation sequencing. We analyzed the significance of TRAIL-R3 upregulation in HBV infection using human hepatocyte transplanted mice and HepG2 cell lines. RESULTS: TRAIL-R3 induction by HBV infection was verified by in vitro and in vivo HBV replication models, and induction was inhibited by antiviral nucleot(s)ide analogue treatment. TRAIL-R3 transcription was regulated by the TRAIL-R3 promoter at -969 to -479 nucleotides upstream from the transcription start site, and by hepatitis B x (HBx) via activation of nuclear factor-κB (NF-κB) signal. TRAIL not only induced cell apoptosis but also inhibited HBV replication. TRAIL-R3 upregulation could inhibit both TRAIL-dependent apoptosis in HBV-infected hepatocytes and TRAIL-mediated suppression of HBV replication. CONCLUSIONS: These results suggest a mechanism by which HBV persists by escaping host immunity through upregulation of TRAIL-R3. Development of novel drugs to inhibit this escape system might lead to complete HBV elimination from human hepatocytes.

The Amino Acid at Position 95 in the Matrix Protein of Rabies Virus Is Involved in Antiviral Stress Granule Formation in Infected Cells.

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

Autoimmune disorders associated with gain of function of the intracellular sensor MDA5.

MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5. These findings provide insight into the association between disorders of the innate immune system and autoimmunity.

Antiviral activity of human OASL protein is mediated by enhancing signaling of the RIG-I RNA sensor.

Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN) and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone-marrow-derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL, Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Trapa bispinosa Roxb. Pericarp Extract Exerts 5α-Reductase Inhibitory Activity in Castrated Benign Prostatic Hyperplasia Model Mice.

Trapa bispinosa Roxb. pericarp extract (TBE) has a polyphenol-rich composition and exhibits potent antioxidant and anti-glycation activities in vitro. In the present study, we investigated the inhibitory effects of TBE on 5α-reductase in vitro using LNCaP cells and in vivo using a mouse model of castrated benign prostatic hyperplasia. TBE showed concentration-dependent inhibitory effects in the 5α-reductase (5αR) activity assay. In a reporter assay using AR-Luc/LNCaP cells, TBE inhibited the activity induced by testosterone, but not that induced by dihydrotestosterone. TBE also suppressed prostate cell proliferation, prostate-specific antigens, and transmembrane protease serine 2 expression in a castrated benign prostatic hyperplasia mouse model. In addition, ellagic acid, but not gallic acid, decreased 5αR and AR-Luc activities. Together, these results suggest a potential role for TBE in benign prostatic hyperplasia through inhibition of 5αR.

Attenuation of regulatory T cell function by type I IFN signaling in an MDA5 gain-of-function mutant mouse model.

Melanoma differentiation-associated gene 5 (MDA5) is an essential viral double-stranded RNA sensor to trigger antiviral immune responses, including type I interferon (IFN) induction. Aberrant activation of this viral sensor is known to cause autoimmune diseases designated as type I interferonopathies. However, the cell types responsible for these diseases and the molecular mechanisms behind their onset and development are still largely unknown. In this study, we revealed the attenuation of regulatory T cell (Treg) function by type I IFN signaling in a mouse model expressing a gain-of-function MDA5 G821S mutant. We found that experimental colitis induced by adoptive transfer of naïve T cells in Rag2-/- mice was rescued by simultaneous transfer of Tregs from wild-type but not from the MDA5 mutant mice. Type I IFN receptor deficiency in the MDA5 mutant mice recovered the suppressive function of MDA5 mutant Tregs. These results suggest that constitutive MDA5 and type I IFN signaling in Tregs decreases the suppressive function of Tregs, potentially contributing to the onset and exacerbation of autoimmune disorders in interferonopathies.

Efficacy and impact of SARS-CoV-2 vaccination on cancer treatment for breast cancer patients: a multi-center prospective observational study.

PURPOSE: Vaccination is an essential strategy to prevent infection in the SARS-CoV-2 pandemic. However, there are concerns about vaccine efficacy and the impact of vaccination on cancer treatment. Additionally, the emergence of novel variants may affect vaccination efficacy. This multi-center, prospective, observational study investigated the efficacy and impact of vaccination against SARS-CoV-2 variants on treatment among breast cancer patients in Japan. METHODS: Patients with breast cancer scheduled to be vaccinated with the SARS-CoV-2 vaccine from May to November 2021 were prospectively enrolled (UMIN000045527). They were stratified into five groups according to their cancer treatment: no treatment, hormone therapy, anti-human epidermal growth factor receptor (HER)2 therapy, chemotherapy, and cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Serum samples for assessing serological responses were collected before the first vaccination and after the second vaccination. RESULTS: Eighty-five breast cancer patients were included. The overall seroconversion rate after second vaccination was 95.3% and the lowest seroconversion rate was 81.8% in the patients under chemotherapy. The overall positivity rate of neutralizing antibodies against the wild-type, α, Δ, κ, and omicron variants were 90.2%, 81.7%, 96.3%, 84.1%, and 8.5%, respectively. Among the patients under chemotherapy or CDK4/6 inhibitors, various degrees of decreased neutralizing antibody titers against SARS-CoV-2 variants were observed. Withdrawal or reduction of systemic therapy because of vaccination was observed in only one patient. CONCLUSION: Our data support SARS-CoV-2 vaccination for breast cancer patients. However, a reduction in neutralizing antibody titers was suggested during chemotherapy and CDK4/6 inhibitors, raising concerns about the impact on long-term infection prevention.

Intracellular virus sensor MDA5 mutation develops autoimmune myocarditis and nephritis.

Mutations in IFIH1 gene encoding viral RNA sensor MDA5 have been reported responsible for many interferonopathies, including Aicardi-Goutières syndrome (AGS) and monogenic lupus, however, the pathological link between IFIH1 mutations and various autoimmune symptoms remains unclear. Here, we generated transgenic mice expressing human MDA5 R779H mutant (R779H Tg), reported in AGS and monogenic lupus patient. Mice spontaneously developed myocarditis and nephritis with upregulation of type I IFNs in the major organs. R779H Tg Mavs-/- and R779H Tg Ifnar-/- showed no phenotypes, indicating direct MDA5-signaling pathway involvement. Rag-2 deficiency and bone marrow cells transfer from wild type to adult mice did not prevent myocarditis development, while mice with cardiomyocyte-specific expression of hMDA5 R779H showed cardiomegaly and high expression of inflammatory cytokines. Taken together, our study clarifies that type I IFNs production and chemokines from cardiomyocytes starts in neonatal period and is critical for the development of myocarditis. Activated lymphocytes and auto-antibodies exacerbate the pathogenesis but are dispensable for the onset.

Psoriasis-like skin disorder in transgenic mice expressing a RIG-I Singleton-Merten syndrome variant.

Mutations in DDX58 (DExD/H-box helicase 58), which encodes the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I), were recently identified in the rare autoimmune disease Singleton-Merten syndrome (SMS). We report the spontaneous development of psoriasis-like skin lesions as an SMS-like symptom in transgenic mice harboring one of the RIG-I SMS variants, E373A. Histological analysis revealed typical characteristics of psoriasis, including the abnormal proliferation and differentiation of keratinocytes leading to epidermal hyperplasia, and infiltrates consisting of neutrophils, dendritic cells and T cells. Levels of the IL-23/IL-17 immune axis cytokines were high in the skin lesions. Rag2-/- transgenic mice showed partial amelioration of the phenotype, with down-regulation of inflammatory cytokines, including IL-17A, suggesting the importance of lymphocytes for the pathogenesis similar to that of human psoriasis. Of note, IL-17A deficiency abolished the skin phenotype, and treatment using the JAK inhibitor tofacitinib not only prevented onset, but also improved the skin manifestations even after onset. Our study provides further evidence for the involvement of RIG-I activation in the onset and progression of psoriasis via type I interferon signaling and the IL-23/IL-17 axis.

Aicardi-Goutières syndrome-like encephalitis in mutant mice with constitutively active MDA5.

MDA5 is a cytoplasmic sensor of viral RNA, triggering type I interferon (IFN-I) production. Constitutively active MDA5 has been linked to autoimmune diseases such as systemic lupus erythematosus, Singleton-Merten syndrome (SMS) and Aicardi-Goutières syndrome (AGS), a genetically determined inflammatory encephalopathy. However, AGS research is challenging due to the lack of animal models. We previously reported lupus-like nephritis and SMS-like bone abnormalities in adult mice with constitutively active MDA5 (Ifih1G821S/+), and herein demonstrate that these mice also exhibit high lethality and spontaneous encephalitis with high IFN-I production during the early postnatal period. Increases in the number of microglia were observed in MDA5/MAVS signaling- and IFN-I-dependent manners. Furthermore, microglia showed an activated state with an increased phagocytic capability and reduced expression of neurotrophic factors. Although multiple auto-antibodies including lupus-related ones were detected in the sera of the mice as well as AGS patients, Ifih1G821S/+Rag2-/- mice also exhibited up-regulation of IFN-I, astrogliosis and microgliosis, indicating that auto-antibodies or lymphocytes are not required for the development of the encephalitis. The IFN-I signature without lymphocytic infiltration observed in Ifih1G821S/+ mice is a typical feature of AGS. Collectively, our results suggest that the Ifih1G821S/+ mice are a model recapitulating AGS and that microglia are a potential target for AGS therapy.

Adsorptive Inhibition of Enveloped Viruses and Nonenveloped Cardioviruses by Antiviral Lignin Produced from Sugarcane Bagasse via Microwave Glycerolysis.

Antiviral lignin was produced by acidic microwave glycerolysis of sugarcane bagasse. The lignin exhibited antiviral activity against nonenveloped (encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelitis virus (TMEV)) and enveloped (vesicular stomatitis virus (VSV), Sindbis virus (SINV), and Newcastle disease virus (NDV)) viruses. A series of lignins with different antiviral activities were prepared by reacting bagasse at 140, 160, 180, and 200 °C to analyze the antiviral mechanism. No difference in ζ-potential was observed among the lignin preparations; however, the lignin prepared at 200 °C (FR200) showed the strongest anti-EMCV activity, smallest hydrodynamic diameter, highest hydrophilicity, and highest affinity for EMCV. FR200 inhibited viral propagation through contact with the virion at the attachment stage to host cells, and the EMCV RNA was intact after treatment. Therefore, the lignin inhibits viral entry to host cells through interactions with the capsid surface. The nonvolatile antiviral substance is potentially useful for preventing the spread of viruses in human living and livestock breeding environments.

Effect of a multimodal non-pharmacological intervention on older people with dementia: a single-case experimental design study.

BACKGROUND: Older people with dementia (PWD) in nursing homes (NHs) tend to have decreased cognitive function, which may cause behavioral and psychological symptoms of dementia (BPSDs) and hinder activities of daily living (ADLs). Therefore, taking measures against the cognitive decline of PWD in NH and, in turn, the decline of BPSDs and ADLs is crucial. The purpose of this study was to test whether a multimodal non-pharmacological intervention (MNPI) is effective in maintaining and improving global cognitive function, BPSDs, and ADLs in PWD in NHs. METHODS: An intervention study using a single-case AB design was conducted in three subjects in NHs. During the non-intervention phase, participants underwent follow-up assessments, and during the intervention phase, they participated in an MNPI. The ABC Dementia Scale (which concurrently assesses ADLs ["A"], BPSDs ["B"], and cognitive function ["C"]) was used for the assessment. RESULTS: One of the three patients showed improvement in dementia severity, global cognitive function, ADLs, and BPSDs. However, the other two participants showed no improvement following the MNPI, although the possibility of a maintenance effect remained. CONCLUSION: Although there is room for improvement of the MNPI, it may be effective in maintaining and improving cognitive function, ADLs, and BPSD, in PWD in NHs. TRIAL REGISTRATION: The University Hospital Medical Information Network Clinical Trials Registry ( http://www.umin.ac.jp/ , No. UMIN000045858, registration date: November 1, 2021).

Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes.

Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-ß induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.

Characterization of distinct molecular interactions responsible for IRF3 and IRF7 phosphorylation and subsequent dimerization.

IRF3 and IRF7 are critical transcription factors in the innate immune response. Their activation is controlled by phosphorylation events, leading to the formation of homodimers that are transcriptionally active. Phosphorylation occurs when IRF3 is recruited to adaptor proteins via a positively charged surface within the regulatory domain of IRF3. This positively charged surface also plays a crucial role in forming the active homodimer by interacting with the phosphorylated sites stabilizing the homodimer. Here, we describe a distinct molecular interaction that is responsible for adaptor docking and hence phosphorylation as well as a separate interaction responsible for the formation of active homodimer. We then demonstrate that IRF7 can be activated by both MAVS and STING in a manner highly similar to that of IRF3 but with one key difference. Regulation of IRF7 appears more tightly controlled; while a single phosphorylation event is sufficient to activate IRF3, at least two phosphorylation events are required for IRF7 activation.

Low RAI2 expression is a marker of poor prognosis in breast cancer.

PURPOSE: Retinoic acid-induced 2 (RAI2) has been shown to be a putative suppressor of the early hematogenous dissemination of tumor cells to the bone marrow in breast cancer. Here, we investigated the associations of RAI2 mRNA and protein expression with clinicopathological factors and prognosis in breast cancer patients with long-term follow-up. METHODS: Invasive breast cancer tissues (n = 604) were analyzed for RAI2 mRNA expression. We examined the associations of clinicopathological factors with the expression levels of RAI2 mRNA in these samples. We also analyzed RAI2 protein expression by immunohistochemistry in invasive breast cancer tissues (n = 422). RESULTS: We identified significant positive associations between low expression of RAI2 mRNA and shorter disease-free survival (DFS), breast-cancer-specific survival (BCSS), and overall survival (OS) in breast cancer patients. We also identified significant positive associations between negative for RAI2 protein expression and shorter DFS, BCSS, and OS in breast cancer patients. Low RAI2 mRNA and negative for RAI2 protein expression were positively associated with larger tumor size, higher tumor grade, and ERα-negativity. Multivariate analyses indicated that not only RAI2 mRNA but also RAI2 protein expression were independent risk factors for both DFS and BCSS in breast cancer patients. The median follow-up periods were 10.3 and 9.3 years for the RAI2 mRNA and protein expression analyses, respectively. CONCLUSIONS: Our findings suggest that RAI2 has a role in the metastasis of breast cancer, and that RAI2 expression could be a promising candidate biomarker of prognosis in breast cancer patients.