ABSTRACT
As a member of interferon regulatory factor family (IRF),IRF3 plays an important role in triggering the expression of type Ⅰ interferons and downstream interferon-stimulated genes,contributing to many biological process.Researches have found that it plays an anti-oncogene role in inhibiting tumor proliferation and migration,inducing cell apoptosis.The mechanism involves in tumor immunity and inflammatory reaction,apoptosis and epithelial mesenchymal transition.The alternative splicing isoforms of IRF3 act as negative modulators of IRF3 and affect tumor development progress.The recent signaling pathways and pathogenesis researches provide new ideas for early diagnosis and treatment of cancer.
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Objective To observe the nuclear translocation of transcription factor NF-κB and IRF-3 in TLR4 silenced EVC304 cells infected by HTNV and to provide new information for anti-HTNV innate immunity and its signal transduction. Methods TLR4~- cells and TLR4~+ cells were infected by HTNV 76-118, respectively. The cells stimulated by LPS were selected as positive control groups, and the cells without stimulation were selected as negative control groups. After 6 hours, indirect immunofluorescence assay(IFA) was used to detect the nuclear translocation of NF-κB and IRF-3. Results The transcription factor NF-κB and IRF-3 transfered into nuclear 6 hours after stimulated by HTNV 76-118. Conclusion TLR4 may mediate the nuclear translocation of transcription factor NF-κB and IRF-3 in HTNV infected human umbilical vein endothelial cells.
ABSTRACT
ays revealed that the-(167-111)bp region Was the minimal promoter of the human IRF3 gene.These results suggested that transcriptional factors such ag E2F might be involved in the transcriptional regulation of IRF3 gene.
ABSTRACT
Objective To construct a luciferase reporter plasmid containing interferon regulatory factor 3(IRF-3)human gene promoter and to evaluate promoter activity in human embryonic kidney(HEK)-293 cells.Methods The 1 000 bp fragment was amplified by PCR with human genomic DNA as a template and was directionally cloned into pGL3-basic multiple cloning sites to construct the luciferase repor-ter plasmid pGL3-pIRF-3.Transfection of HEK-293 cells with the promoter-driven lucife-rase construct was performed to induce lucife-rase gene expression and calculate the relative luciferase activity unit(RLU).Promoter sequence of 1 000 bp upstream of transcription initiation site of IRF-3 was analyzed by using Promoter 2.0 Prediction software.Results DNA sequencing and restriction endonuclease analysis verified the successful construction of the plasmid pGL3-pIRF-3.This IRF-3 promoter exhibited a strong promoter activity with an increase of 42.2-fold of RLU in HEK-293 cells when compared with pGL-3 basic vector.The transfection experiment confirmed that the levels of its activation were significantly higher than that in controls in HEK-293 cells.Function analysis of IRF-3 promoter disclosed seve-ral GATA-1 and specific protein 1(Sp1) sites and E2F in minimal promoter region.Conclusion The plasmid pGL3-pIRF-3 promoter is successfully constructed and has a strong basal promoter activity in HEK-293 cells.
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Objective To explore the inhibitory effect of mutant influenza A viruses to the activation of interferon regulatory factor 3 (IRF-3). Methods HEK293 cells were infected with A/FM/1/47,A/HK/1/68, A/HK/1/68-MA20, A/HK/1/68-MA20C and positive control Sendai virus (SV). Whether the slowly moved phosphorylation form Ⅲ and Ⅳ of IRF-3 appeared or not was compared by Western blot in cells infected with these viruses. Wild type of NS1 from A/HK/1/68 and mutant NS1 from A/HK/1/68-MA20 were subcloned into pcDNA3.1-flag respectively. They were transfected in HEK 293 cells respectively. At 16 hours posttransfection, cells were infected with Sendai virus for 8 hours. Whole cell extracts were analyzed by Western blot and then probed with monoclonal flag antibody to check the expression of NS1, or with anti-IRF-3 to observe the inhibitory effects of the wild and mutant NS1 to the activated IRF-3. Luciferase assay was carried out by co-transfection with reporter plasmid, pGL2B with interferon ? promoter, and wild or mutant NS1 cDNA expression plasmid. SV was used to infect these cells after the co-transfection. Results Only less virulent A/HK/1/68-MA20 and positive control SV can activate IRF-3. Activated form Ⅲ and Ⅳ of IRF-3 began to appear 9 hours post infection (h.p.i), and most significant activated IRF-3 appeared 23 and 26 h.p.i. Sequence analysis of NS1 of MA20 revealed that nucleotide position number 94 is mutated from T to C, and amino acid at position number 23 is changed from valine to alanine. Co-transfected with wild type NS1 made form Ⅲ and Ⅳ of IRF-3 almost disappear, but not mutant NS1. In the luciferase functional analysis, wild type NS1 can inhibit the luciferase activity of IFN-? promoter, which was induced by SV, to around 1/10. Again no inhibitory effects was observed of mutant NS1 in the luciferase assay. Conclusion The mechanism that A/HK/1/68-MA20 can activate IRF-3 is that point mutant NS1 abolished the inhibitory function of NS1.