Rig-I regulates NF-κB activity through binding to Nf-κb1 3'-UTR mRNA.

Retinoic acid inducible gene I (RIG-I) senses viral RNAs and triggers innate antiviral responses through induction of type I IFNs and inflammatory cytokines. However, whether RIG-I interacts with host cellular RNA remains undetermined. Here we report that Rig-I interacts with multiple cellular mRNAs, especially Nf-κb1. Rig-I is required for NF-κB activity via regulating Nf-κb1 expression at posttranscriptional levels. It interacts with the multiple binding sites within 3'-UTR of Nf-κb1 mRNA. Further analyses reveal that three distinct tandem motifs enriched in the 3'-UTR fragments can be recognized by Rig-I. The 3'-UTR binding with Rig-I plays a critical role in normal translation of Nf-κb1 by recruiting the ribosomal proteins [ribosomal protein L13 (Rpl13) and Rpl8] and rRNAs (18S and 28S). Down-regulation of Rig-I or Rpl13 significantly reduces Nf-κb1 and 3'-UTR-mediated luciferase expression levels. These findings indicate that Rig-I functions as a positive regulator for NF-κB signaling and is involved in multiple biological processes in addition to host antivirus immunity.

RIG-I plays a critical role in negatively regulating granulocytic proliferation.

RIG-I has been implicated in innate immunity by sensing intracellular viral RNAs and inducing type I IFN production. However, we have found a significant RIG-I induction in a biological setting without active viral infection-namely, during RA-induced terminal granulocytic differentiation of acute myeloid leukemias. Here, we present evidence that a significant Rig-I induction also occurs during normal myelopoiesis and that the disruption of the Rig-I gene in mice leads to the development of a progressive myeloproliferative disorder. The initiation of progressive myeloproliferative disorder is mainly due to an intrinsic defect of Rig-I(-/-) myeloid cells, which are characterized by a reduced expression of IFN consensus sequence binding protein, a major regulator of myeloid differentiation. Thus, our study reveals a critical regulatory role of Rig-I in modulating the generation and differentiation of granulocytes.

[Establishment of transgenic mice for HRX-EEN fusion gene].

OBJECTIVE: To study the biological function of fusion gene HRX-EEN and its role in leukemogenesis, and to provide an ideal animal model for anti-leukemia drug screening. METHODS: HRX-EEN fusion gene was constructed by use of three different DNA fragments, and it was inserted into hCG transgenic vector. G(0) transgenic mice were obtained by microinjection of the recombined DNA into the pronucleus of zygotes, followed by implantation of the injected zygotes into pseudopregnant mice. The integration of the transgene was tested by PCR and its expression by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The sequence of recombined HRX-EEN gene was confirmed by sequencing. PCR testing revealed a total of 7 G(0) transgenic mice, these mice were then mated with C57 wild type mice. Except mouse No. 35 that died, the others all had their F1 offsprings. From these 6 lines of transgenic mice, HRX-EEN gene was found to be stably expressed in 3 lines by RT-PCR. Up to now, all transgenic mice expressing the fusion gene have no obvious abnormal phenotypes. CONCLUSION: A transgenic mice model in which the HRX-EEN fusion gene can be stably expressed has been established.

Emodin enhances sensitivity of gallbladder cancer cells to platinum drugs via glutathion depletion and MRP1 downregulation.

Glutathione conjugation and transportation of glutathione conjugates of anticancer drugs out of cells are important for detoxification of many anticancer drugs. Inhibition of this detoxification system has recently been proposed as a strategy to treat drug-resistant solid tumors. Gallbladder carcinoma is resistant to many anticancer drugs, therefore, it is needed to develop a novel strategy for cancer therapy. In the present study, we tested the effect of emodin (1,3,8-trihydroxy-6-methylanthraquinone), a reactive oxygen species (ROS) generator reported by our group previously, in combination with cisplatin (CDDP), carboplatin (CBP) or oxaliplatin in treating the gallbladder carcinoma cell line SGC996. Our results showed that co-treatment with emodin could remarkably enhance chemosensitivity of SGC996 cells in comparison with cisplatin, carboplatin or oxaliplatin treatment alone. We found that the mechanisms may be attributed to reduction of glutathione level, and downregulation of multidrug resistance-related protein 1 (MRP1) expression in SGC996 cells. The experiments on tumor-bearing mice showed that emodin/cisplatin co-treatment inhibited the tumor growth in vivo via increasing tumor cell apoptosis and downregulating MRP1 expression. In conclusion, emodin can work as an adjunct to enhance the anticancer effect of platinum drugs in gallbladder cancer cells via ROS-related mechanisms.

An essential role for RIG-I in toll-like receptor-stimulated phagocytosis.

Retinoic acid-inducible gene-I (RIG-I) plays an important role in antiviral response by recognizing double-stranded RNA. Here we demonstrate an unanticipated role of RIG-I in Toll-like receptor (TLR)-stimulated phagocytosis. Stimulation with lipopolysaccharide (LPS), a ligand of TLR4, induced the expression of RIG-I in macrophages. Depletion of RIG-I by RNAi or gene targeting inhibited the LPS-induced phagocytosis of bacteria. Cellular processes involved in phagocytosis, such as small GTPase Cdc42/Rac1 activation, actin polymerization, and actin-regulator Arp2/3 recruitment, were also impaired in RIG-I-deficient macrophages activated by LPS. Moreover, RIG-I(-/-) mice were found to be more susceptible to infection with Escherichia coli as compared to wild-type mice. Thus, the regulatory functions of RIG-I are strikingly broad, including a role not only in antiviral responses but in antibacterial responses as well.

Rig-I-/- mice develop colitis associated with downregulation of G alpha i2.

RIG-I (retinoid acid-inducible gene-I), a putative RNA helicase with a cytoplasmic caspase-recruitment domain (CARD), was identified as a pattern-recognition receptor (PRR) that mediates antiviral immunity by inducing type I interferon production. To further study the biological function of RIG-I, we generated Rig-I(-/-) mice through homologous recombination, taking a different strategy to the previously reported strategy. Our Rig-I(-/-) mice are viable and fertile. Histological analysis shows that Rig-I(-/-) mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate sodium-induced colitis. Accordingly, the size and number of Peyer's patches dramatically decreased in mutant mice. The peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naive T cells, indicating an important role for Rig-I in the regulation of T-cell activation. It was further found that Rig-I deficiency leads to the downregulation of G protein alpha i2 subunit (G alpha i2) in various tissues, including T and B lymphocytes. By contrast, upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated G alpha i2 expression. Moreover, G alpha i2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these findings suggest that Rig-I has crucial roles in the regulation of G alpha i2 expression and T-cell activation. The development of colitis may be, at least in part, associated with downregulation of G alpha i2 and disturbed T-cell homeostasis.

[HUP98-HOXA9 transgenic mice are susceptible to N-ethyl-N-nitrosourea stimulation in leukemogenesis].

OBJECTIVE: In order to investigate the leukemogenic potential of NUP98-HOXA9 fusion gene in vivo. METHODS: Molecular cloning technology was used to construct NUP98-HOXA9 transgenic plasmid and NUP98-HOXA9 transgenic mice were generated. The genotype and phenotype of the NUP98-HOXA9 transgenic mice were analyzed by PCR, RT-PCR and colony-forming assay. The effect of N-ethyl-N-nitrosourea (ENU) stimulation on the transgenic mice was analyzed by peripheral blood count, bone marrow (BM) cells morphology pathological examination. RESULTS: The transgenic expression was detected in 5 independent lines of NUP98-HOXA9 transgenic mice, but no expected phenotypes was found in 2 year follow-up. Upon ENU stimulation, 2 of 10 transgenic mice developed myeloid leukemia, suggesting that NUP98-HOXA9 transgenic mice have increased susceptibility to ENU mutagenesis in leukemogenesis. CONCLUSION: The fusion gene expressed in BM cells of NUP98-HOXA9 transgenic mice. It seems that the expression of the fusion gene is insufficient to trigger leukemogenesis. However, the increased susceptibility to ENU mutagenesis suggests that NUP98-HOXA9 fusion gene might play a potential role in leukemogenesis.

[Development of human myeloid leukemia-like phenotype in NUP98-PMX1 transgenic mice].

OBJECTIVE: In order to investigate the leukemogenic potential of NUP98-PMX1 fusion gene in vivo. METHODS: NUP98-PMX1 transgenic mice were generated, in which the fusion gene was driven by hCG promoter and expressed in myeloid cells at early stage of differentiation. Molecular cloning technology was used to construct NUP98-PMX1 transgenic plasmid. The genotype and phenotype of the NUP98-PMX1 transgenic mice were analyzed by PCR, RT-PCR, peripheral blood count (PBC), bone marrow (BM) cells morphology and pathological examination. RESULTS: NIH3T3 cells transfected with NUP98-PMX1 fusion gene grew faster, formed colonies in soft agar, and developed tumors in 10 inoculated nude mice. Among 8 disordered NUP98-PMX1 transgenic mice, 4 developed myeloid leukemia-like phenotype, including 3 resembling human chronic myeloid leukemia. CONCLUSION: NUP98-PMX1 has oncogenic activity and plays a crucial role in leukemogenesis.