Objective To investigate the effect of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) on the proliferation, migration and tumor immune microenvironment of colorectal cancer cells and its possible molecular mechanism. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of IGF2BP2 and MYC in colorectal cancer and adjacent tissues. The expression of IGF2BP2 in HCT-116 and SW480 human colorectal cancer cells was silenced by RNA interference (RNAi), and the silencing effect was detected by quantitative real-time PCR. After knocking down IGF2BP2, colony formation assay, CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were employed to detect cell colony formation and proliferation ability. Transwell^TM assay was used to detect cell migration ability. Quantitative real-time PCR was used to detect the mRNA expression of IGF2BP2, MYC, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10). The protein expression of IGF2BP2 and MYC was detected by western blot. The binding ability of IGF2BP2 and MYC in HCT-116 cells was detected by quantitative real-time PCR after RNA immunoprecipitation. Results The results of TCGA database showed that the expression of IGF2BP2 and MYC in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the survival time of colorectal cancer patients with high expression of IGF2BP2 was shorter. After silencing IGF2BP2, the viability, proliferation and migration of HCT-116 and SW480 cells were decreased. The mRNA expression of MYC, TGF-β and IL-10 in IGF2BP2 knockdown group was significantly decreased, while the expression of TNF-α mRNA was increased. The expression of MYC protein and the stability of MYC mRNA were significantly decreased. RIP-qPCR results showed that IGF2BP2 could bind to MYC mRNA. Conclusion Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression.
Humans ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Colorectal Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic ; Interleukin-10/metabolism* ; RNA, Messenger ; RNA-Binding Proteins/metabolism* ; Transforming Growth Factor beta/genetics* ; Tumor Microenvironment/immunology* ; Tumor Necrosis Factor-alpha/metabolism* ; Proto-Oncogene Proteins c-myc/metabolism*

Nucleoprotein (NP) of influenza virus is highly conserved and type-specific. NP can trigger strong cell-mediated immune responses in host and is involved in the protection against the challenges with different subtype influenza viruses. Here, NP of an avian H5N1 (A/Hubei/1/2010, HB) was expressed by baculovirus surface-display technology and its immunogenicity as well as protective mechanism was investigated in mice infection model. Western blot and immunolabeled electron microscopy assay showed NP was displayed on baculovirus surface. ELISA results showed NP could induce high level of anti-NP IgG in the sera from NP-Bac-inoculated mice. Two cellular immune peptides (NP57-74 IQNSITIERMVLSAFDER and NP441-458 RTEIIKMMESARPEDLSF) were identified by IFN-gamma ELISPOT assay. NP57-66 and NP441-450 and NP protein could be able to trigger the activation of CD4+ and CD8+ T cells, and the response of CD8+ T was more predominant. The challenge study of mice-adapted virus A/PR/8/34 (H1N1) showed that NP-Bac could reduce viral load and attenuate the damage to lung tissue. 50% protection ratio against the virus could be detected.
Animals ; Antibodies, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Cross Protection ; Enzyme-Linked Immunospot Assay ; Female ; Humans ; Immunity, Cellular ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; RNA-Binding Proteins ; genetics ; immunology ; T-Lymphocytes ; immunology ; Viral Core Proteins ; genetics ; immunology

Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell. Amongst the spillage, the secondary messenger molecule, cyclic dimeric guanosine monophosphate (c di-GMP), was recently shown to be bound by stimulator of interferon genes (STING). Binding of c di-GMP by STING activates the Tank Binding Kinase (TBK1) mediated signaling cascades that galvanize the body's defenses for elimination of the pathogen. In addition to c di-GMP, STING has also been shown to function in innate immune responses against pathogen associated molecular patterns (PAMPs) originating from the DNA or RNA of pathogens. The pivotal role of STING in host defense is exemplified by the fact that STING(-/-) mice die upon infection by HSV-1. Thus, STING plays an essential role in innate immune responses against pathogens. This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the immune defenses against invading microbes. Similarly, STING could be targeted for mitigating the inflammatory responses augmented by the innate immune system. This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in delineating the mechanism of STING-mediated responses.
Animals ; Cyclic GMP ; physiology ; Dimerization ; Herpes Simplex ; immunology ; pathology ; Humans ; Immunity, Innate ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Protein Binding ; RNA, Viral ; metabolism ; STAT6 Transcription Factor ; metabolism ; Second Messenger Systems

Interferons play critical roles in tumor pathogenesis by controlling apoptosis and through cellular anti-proliferative and differentiation activities. Interferon inducible transmembrane protein (IFITM) family genes have been implicated in several cellular processes such as the homotypic cell adhesion functions of IFN and cellular anti-proliferative activities. Expression levels of IFITM genes have been found to be up-regulated in gastric cancer cells and colorectal tumors. IFITM3 (also known as 1-8U) is a member of the IFITM family, and has been described as a key player in specification of germ cell fate. IFITM3 was first isolated from a genetic screen aimed at identifying genes involved in acquisition of germ cell competence. It has been proposed that epiblast cells have the highest expression of IFITM3 initiated germ cell specification and that homotypic association can discriminate germ cells from their somatic neighbors. In an attempt to better understand the genetic influences of IFITM3 on ulcerative colitis, we have identified possible variation sites and single nucleotide polymorphisms (SNPs) through two exons and their boundary IFITM3 intron sequences including the ~2.1 kb promoter regions. To determine whether or not these IFITM3 SNPs are associated with susceptibility to ulcerative colitis, frequencies of the genotype and allele of IFITM3 polymorphisms were analyzed on genomic DNAs isolated from patients with ulcerative colitis and from healthy controls. We also investigated the haplotype frequencies constructed by these SNPs in both groups. In this study, we also showed that expression level of IFITM3 mRNA was significantly higher in tissues of the ileum and cecum of the digestive system. We identified a total of seven SNPs and multiple variation regions in the IFITM3 gene. The genotype frequency of the g.-204T>G polymorphism in patients with ulcerative colitis was significantly different from that of the control group. Our results strongly suggest that polymorphisms of the IFITM3 gene may be associated with susceptibility to ulcerative colitis.
Cecum/*metabolism ; Colitis, Ulcerative/epidemiology/*genetics/immunology ; Gene Expression Profiling ; Gene Frequency ; Genetic Association Studies ; Genetic Predisposition to Disease ; Haplotypes ; Ileum/*metabolism ; Korea ; Membrane Proteins/*genetics/immunology/metabolism ; Organ Specificity ; Polymorphism, Single Nucleotide ; RNA-Binding Proteins/*genetics/immunology/metabolism

Chemokines and chemokine receptors play a role in migration of circulating leukocytes to the region of inflammation. Human LZIP is an uncharacterized transcription factor and is known to participate in leukotactin (Lkn)-1/CCL15-induced cell migration. We investigated the role of human LZIP in expression of CC chemokine receptors (CCRs) and its involvement in monocyte migration. RNase protection analysis showed that LZIP increased mRNA expression of CCR2 and CCR1 in THP-1 cells. Surface expressions of both CCR2 and CCR1 were also increased by LZIP. Results from an electrophoretic mobility shift assay showed that LZIP binds to the C/EBP element in the CCR2 promoter. LZIP also enhanced the chemotactic activities of monocyte chemoattractant protein-1/CCL2 and Lkn-1. These results suggest that LZIP regulates expression of chemokine receptors that are involved in monocyte migration.
Atherosclerosis/drug therapy/etiology ; CCAAT-Enhancer-Binding Proteins/genetics/immunology/*metabolism ; Cell Line, Tumor ; Cell Movement/drug effects/*physiology ; Chemokine CCL2/*pharmacology ; Chemokines, CC/pharmacology ; Cyclic AMP Response Element-Binding ; Humans ; Macrophage Inflammatory Proteins/pharmacology ; Monocytes/drug effects/metabolism ; Promoter Regions, Genetic ; Protein Binding ; RNA, Messenger/analysis ; Receptors, CCR1/biosynthesis/genetics ; Receptors, CCR2/*biosynthesis/genetics ; Transcriptional Activation/drug effects ; Transfection ; Transgenes

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta , IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta??(3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.
ATP-Binding Cassette Transporters/*genetics ; Blotting, Western ; Cells, Cultured ; Endothelial Cells/metabolism/*virology ; GTP-Binding Proteins/*genetics ; *Gene Expression Regulation ; Hantaan virus/*immunology ; Histocompatibility Antigens Class I/analysis ; Humans ; Interferon Regulatory Factor-3/genetics ; Interferon Regulatory Factor-7/*genetics ; Interferons/*genetics ; RNA, Messenger/analysis

BACKGROUNDSam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line.

METHODSBy using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometric analyses were performed to investigate the Sam68 functions.

RESULTSA slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells.

CONCLUSIONSThe proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; physiology ; Animals ; B-Lymphocytes ; cytology ; immunology ; physiology ; Binding Sites ; genetics ; Blotting, Western ; Cell Cycle ; physiology ; Cell Death ; physiology ; Cell Growth Processes ; drug effects ; physiology ; Cell Line, Tumor ; Culture Media, Serum-Free ; pharmacology ; Mutation ; genetics ; Phosphorylation ; Proline ; genetics ; RNA-Binding Proteins ; genetics ; metabolism ; physiology ; Receptors, Antigen, B-Cell ; immunology ; physiology ; Signal Transduction ; drug effects ; physiology ; Tyrosine ; metabolism

This study was aimed to construct nucleic acid vaccine containing the coding region of the CML28 gene and to express it in human dendritic cells. The full length of CML28 cDNA was amplified from K562 by RT-PCR and subcloned into pGEM-T vector. The CML28 fragment was digested and subsequently inserted into the EcoRI-Xba I sites of pcDNA3.1HisA to construct the recombinant expression vector pcDNA3.1HisA-CML28, which was identified by restrition analysis and sequencing. Human dendritic cells (DC) were separated from peripheral blood mononuclear cells (PBMC) by culture with rhGM-CSF, rhIL-4 and assessed by flow cytometry. The constructed plasmid pcDNA3.1 HisA-CML28 was transfected into DC by electroporation. Western blot was used to detect the expression of fusion protein His-CML28. The results showed that recombinant plasmid pcDNA3.1HisA-CML28 contained the correct full CML28 cDNA identified by restriction analysis and sequencing, and can express the fusion protein His-CML28 in DCs. It is concluded that nucleic acid vaccine containing CML28 gene was constructed and expressed in DC successfully.
Antigens, Neoplasm ; genetics ; immunology ; metabolism ; Antigens, Surface ; genetics ; immunology ; metabolism ; Blotting, Western ; Cells, Cultured ; Cloning, Molecular ; DNA, Complementary ; genetics ; Dendritic Cells ; cytology ; immunology ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Exoribonucleases ; genetics ; immunology ; metabolism ; Exosome Multienzyme Ribonuclease Complex ; Flow Cytometry ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; RNA-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection ; methods ; Vaccines, DNA ; biosynthesis ; genetics ; immunology

BACKGROUNDAnterior chamber associated immune deviation (ACAID) is characterized by a Th2 cell response. GATA-3 has been shown to be necessary for the activation of Th2 cells. This study was designed to examine the expression of GATA-3 in the development of ACAID.

METHODSACAID was induced by injection of 50 microg interphotoreceptor retinoid binding protein (IRBP) into the anterior chamber (AC) of Wistar rats. Delayed-type hypersensitivity (DTH) was evaluated on day 3, 7, 14, 21, 28 after IRBP inoculation. GATA-3 expression was detected using immunohistochemical staining. The expression of GATA-3 mRNA at different time points after AC injection of IRBP was assayed by reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTSA significant DTH reaction was observed in Wistar rats on day 3 and 5 after IRBP inoculation. The DTH reaction was decreased 7 days after IRBP inoculation. GATA-3 expression was weak at both mRNA and protein levels in the normal spleen, but was significantly increased on day 5, 7, 14, and 21 after AC injection of IRBP.

CONCLUSIONThe expression of GATA-3 is increased during ACAID, suggesting that GATA-3 may be involved in the development of ACAID.

Animals ; Anterior Chamber ; immunology ; Eye Proteins ; immunology ; Female ; GATA3 Transcription Factor ; analysis ; genetics ; physiology ; Hypersensitivity, Delayed ; immunology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Retinol-Binding Proteins ; immunology ; Spleen ; metabolism ; Th2 Cells ; immunology

OBJECTIVETo better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.

METHODSBy using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining.

RESULTSA hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression.

CONCLUSIONSThe G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Biomarkers, Tumor ; Breast Neoplasms ; metabolism ; pathology ; Carrier Proteins ; genetics ; immunology ; metabolism ; DNA Helicases ; Female ; Genetic Vectors ; Humans ; Hybridomas ; secretion ; Lymphatic Metastasis ; Male ; Mice ; Mice, Inbred BALB C ; Poly-ADP-Ribose Binding Proteins ; RNA Helicases ; RNA Recognition Motif Proteins ; Receptor, ErbB-2 ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Tumor Cells, Cultured