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@#ObjectiveTo investigate the effect of activation of RIG-Ⅰsignaling pathway by leucine rich repeat containing23(LRRC23)on replication of influenza virus.MethodsOverexpression and knock-down of LRRC23 were performed in A549 cells to investigate its effect on influenza virus replication. A549 cells were transfected with pcLRRC23 plasmid or siLRRC23(small interfering LRRC23)for 24 h and then infected with influenza virus A/jingfang/1/86(H1N1). The virus titer and HA protein expression level in the cell supernatant were determined by plaque assay and ELISA respectively.The expression of LRRC23,RIG-Ⅰ,MAVS,M1 and HA at gene and protein levels were determined by qRT-PCR and Western blot respectively. The interactions between LRRC23 and RIG-Ⅰwere analyzed by co-immunopre-cipitation(Co-IP)and immunofluorescence assay(IFA). IFN-β-luc and NF-κB-luc activities were determined by dual-luciferase reporter assay.ResultsThe LRRC23 overexpression significantly decreased the influenza virus titer and inhibited the expression of HA protein in the supernatant of A549 cells,while enhanced the NF-κB and IFNβ activations by activation of RIG-Ⅰ-MAVS signaling pathway,resulting in the inhibition of expressions of M1 gene and HA protein. Conversely,the knock-down of LRRC23 increased the protein expression level of HA in the supernatant of A549 cells,up-regulated the relative expression level of M1 gene and down-regulated those of RIG-ⅠmRNA and MAVS mRNA.ConclusionLRRC23 plays an essential role in innate antiviral response by inhibiting influenza virus replication through activation of RIG-Ⅰsignaling pathway.
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Objective:To investigate the expressions of TRIM25 and RIG-Ⅰ in liver cancer tissues and their relationship with the prognosis of patients.Methods:The data of 82 patients with liver cancer who were admitted to the Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University from January 2017 to January 2018 were retrospectively analyzed, and their cancer tissue and paracancerous tissue (>5 cm from the edge of the tumor) specimens were collected. The protein expressions of TRIM25 and RIG-Ⅰ in cancer tissues and paracancerous tissues were detected by immunohistochemistry. The relationship between TRIM25 and RIG-Ⅰ expressions in cancer tissues of patients and clinicopathological features was analyzed. Kaplan-Meier method was used to analyze the overall survival (OS) of patients with different TRIM25 and RIG-Ⅰ expression status.Results:The positive rate of TRIM25 in cancer tissues was higher than that in paracancerous tissues [68.29% (56/82) vs. 21.95% (18/82), P < 0.001], and the positive rate of RIG-Ⅰ in cancer tissues was lower than that in paracancerous tissues [31.71% (26/82) vs. 74.39% (61/82), P < 0.001]. The positive rate of TRIM25 in cancer tissues of poorly differentiated patients was higher than that of highly and moderately differentiated patients ( P < 0.05), and the positive rate of RIG-Ⅰ was lower than that of highly and moderately differentiated patients ( P < 0.05). The positive rate of TRIM25 in cancer tissues of patients with extrahepatic metastasis was higher than that of patients without extrahepatic metastasis ( P < 0.05), but the positive rate of RIG-Ⅰ was lower than that of patients without extrahepatic metastasis ( P < 0.05). The positive rate of TRIM25 in patients with clinical Ⅲ-Ⅳ was higher than that in patients with stage Ⅰ-Ⅱ ( P < 0.05), but the positive rate of RIG-Ⅰ was lower than that in patients with stage Ⅰ-Ⅱ ( P < 0.05). The median follow-up time was 27 months (4-48 months), 2 patients were lost to follow-up. At the end of follow-up in January 2022, the overall survival rate was 43.75% (35/80). The survival rates of patients with TRIM25-positive and TRIM25-negative cancer tissues were 33.33% (18/54) and 65.38% (17/26), respectively. The survival rates of patients with RIG-Ⅰ-positive and RIG-Ⅰ-negative cancer tissues were 64.00% (16/25) and 34.55% (19/55), respectively. Kaplan-Meier analysis showed that the OS of patients with TRIM25-negative cancer tissues was better than that of patients with TRIM25-positive cancer tissues, and the OS of patients with RIG-Ⅰ-positive cancer tissues was better than that of patients with TRIM25-negative cancer tissues, and the differences were statistically significant (both P < 0.05). Conclusions:The expression of TRIM25 is increased and the expression of RIG-Ⅰ is decreased in liver cancer tissues. The expressions of TRIM25 and RIG-Ⅰ in liver cancer tissues are associated with prognosis.
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Objective:To provide experimental evidences for choosing murine models in the pathogenesis research of thymic impairment induced by viral infection,we compared the impacts of polycytidylic acid(Poly(I:C)) and dexamethasone(DEX) on the thymic morphology and thymic output function,and explored the implication of RLR signaling pathway.Methods: 24 male C57BL/6 mice were randomly assigned into three groups and treated with Poly(I:C),DEX,or saline respectively.Thereafter,their thymic morphology,pathological changes,thymic index,and thymic pathology were examined.Their contents of T-cell receptor excision circles (TRECs) and proportions of the naive CD4+T cell in the peripheral blood were determined to evaluate their thymic output function.The expression levels of thymic RLR/MAVS/IFN-α/β signaling pathway and IL-1β were also measured.Results: Both Poly (I:C) and DEX treatment caused thymic atrophy in appearance and structural destruction under the microscope inspection,and DEX treatment did much more severe damage,especially to the thymic cortex.TRECs decreased significantly in both groups.The proportions of na?ve/memory CD4+T cell subsets remained stable,though total CD4+T cell decreased in DEX group,while the proportion of na?ve CD4+T cell in Poly (I:C) group increased significantly.The expression of RIG-Ⅰ,MDA5,LGP2,and IFN-α/β were up-regulated in DEX group, while it remained unchanged in Poly (I:C) group.Conclusion:Both Poly (I:C) and DEX induced thymic atrophy and the impaired thymic output function.Nevertheless,the expression of RLR-IFN signaling pathway up-regulated more significantly in DEX group instead of in Poly (I:C) group.These results implied the existence of different pathological manifestations and mechanisms underlying the impaired thymic function in different animal models,as well as impact on na?ve/memory CD4+T cell proportions.Our research provides references for choosing animal models in the basic research and drug development for viral infection induced thymic atrophy based on the RLR signaling pathway.
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Objective To study the mechanism of RING finger protein 34 ( RNF34 ) involved in innate immunity . Methods Recombinant PCR was used and transient expression of the plasmid was achieved in HEK 293T cells.The cells were stimulated with Sendai virus ( SeV) or N-RIG-Ⅰfor the indicated time while luciferase activity was observed using the dual-luciferase reporter assay kit .Results We constructed the plasmid pcDNA 3-Flag-RNF34 and its three mutations .The study found that when stimulated by SeV , RNF34 could inhibit the activity of NF-κB and IFN-βmore significantly than RNF34-ΔFYVE, RNF34-ΔCID and RNF34-ΔRING.We also found that RNF 34 and its three mutants had similar inhibitory effect when the activation of NF-κB and IFN-βwas stimulated by the N-RIG-Ⅰ.Conclusion RNF34 negatively regulates innate immunity by acting on the RIG-Ⅰ-MAVS signaling pathway .
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At present, the etiology and pathogenesis of multiple sclerosis are unclear. RIG-Ⅰ-like receptors are a new-ly discovered pattern recognition receptors (RLRs), which are located in cytoplasm. They can recognize the helicase of viral dsRNAs, and interact with interferon beta promoter stimulator (IPS)-1 through their caspase activation recruitment domain (CARD), then form IPS-1 signalsome and induce the expression of interferon typeⅠ(Ⅰ-IFN), thereby initiate innate im-mune response and induce antiviral response. Recent studies have found that mice lacking IPS-1 would develop exacerbated disease and accompanied by markedly higher inflammation, increasing axonal damage and demyelination. Furthermore, initi-ating the RIG-Ⅰ-like helicase receptor on the immune cells can alleviate inflammation and myelin fracture in multiple scle-rosis of mouse model, thus limit the incidence of paralysis. This paper is a review about the research progress on RLRs in the treatment of multiple sclerosis.
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Objective To investigate the expression of pattern recognition receptor (PRR)of immune cells from the patients with enterovirus 71 ( EV 71 ) infection and the changes of cytokines. Methods Seventy-one patients and 20 age-matched healthy children were enrolled in the study. The patients were divided into three groups according to the critical degree: 20 mild patients, 25 severe patients and 26 critical patients. Real-time PCR were used to evaluate the levels of retinoic acidinducible gene Ⅰ ( RIG- Ⅰ ), melanoma differentitation-associated gene 5 ( MDA5 ) and cytokines IL-12, IFN-α mRNA expression in peripheral blood mononuclear cells (PBMC). The proportions of Toll like receptors(TLRs) on monocytes/macrophages (MC) , myloiddentritic cells (mDC) and plasmacytoiddentritic cells (pDC) were analyzed by flow cytometry. The concentrations of plasma cytokines( IL-12, IFN-α) were measured by ELISA. Results ( 1 ) The proportion of TLR7 is the unique TLR which is increased in mild patients and it was significantly elevated in MC, mDC and pDC in severe group (P＜0.05), TLR2, TLR3 and TLR4 also had an enhanced expression in MC and mDC. The enhanced expression of TLRs mentioned above had a decreased trend in critical patients. (2)Transcription levels of RIG- Ⅰ and MDA5 was significantly elevated in EV71 infected children.(3)The expression levels of DC-associated cytokines gene( IL-12 and IFN-α) have an increased trend in mild cases and these cytokines were remarkably increased in severe patients (P＜0.05), whereas decreased in critical cases (P＜0.05). ConclusionTLR7 might be the main PRR recognizing EV71 in immune cells and RIG-Ⅰ/MDA5 might also take part in the recognition of EV71. The exogenous or endogenous ligands released from bacterial infection and cell destruction could result in the activation of TLR2 or TLR4, inducing the inflammatory response.
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RIG-Ⅰ, the abbreviation of retinoic acid inducible gene-Ⅰ, can be induced to express in many type cells by various stimuli. Recently, it was identified as an intracellular regulator for RNA virus-induced antiviral response in innate immunity. Its discovery, expression induction, structure, research status of its function, homologous proteins and functional mechanism etc. were summarized, and its further research pulses are also prospected meanwhile.