This study was purposed to investigate the effect of mutation and single nucleotide polymorphism (SNP) of suppressor of cytokine signaling (SOCS) on the typical myeloproliferative neoplasms (MPN) and its mechanism. The mutation and SNP of SOCS1, SOCS2, SOCS3 genes in 100 MPN patients were detected by RT-PCR and direct sequencing. The results showed that among 100 cases there were 21 cases with A→C polymorphism in the 63th site nucleotide of the 15 SOCS3 exon (SNP library no reported), 18 cases with A→C polymorphism in the 1779th site nucleotide of the 15 SOCS3 exon, 49 cases with A→G polymorphism in the 2249th site nucleotide of the 15 SOCS3 exon (SNP library no reported), 39 cases with T→C polymorphism in the 2366th site nucleotide of the 15 SOCS3 exon (SNP library no reported), 9 cases with T→C polymorphism in the exon of 15 SOCS2 gene (SNP library no reported). SOCS3 SNP was found in patients with significantly advanced age at diagnosis, the leukocyte count and platelet level were higher than those in patients with wild type, JAK2V617 mutations was found in 87.65% SOCS3 SNP. It is concluded that the SOCS may be an important target for anticancer therapy, the single nucleotide polymorphism of SOCS may involve to pathogenesis of MPN.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Exons ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Myeloproliferative Disorders ; genetics ; Polymorphism, Single Nucleotide ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; Young Adult

Suppressor of cytokine signaling (SOCS) is a new family of proteins produced in cells. It may play an important role in classic negative feedback loop to regulate cytokine signal transduction. SOCS-1 was observed and confirmed firstly. Expression of SOCS-1 can inhibit cytokine signal transduction of some cytokines, such as IL-6, LIF, OSM, INF-gamma, GH, and so on, many immune responses are regulated by them in vivo. Abnormal expression of SOCS-1 is closely related to some human diseases. It plays an important role in the development of leukemia, rheumatoid arthritis, liver cirrhosis and liver cancer. In this review, the advances of research on the relationship between SOCS-1 and cytokine, and its correlation with some diseases were summarized.
DNA Methylation ; Humans ; Interleukin-6 ; biosynthesis ; Intracellular Signaling Peptides and Proteins ; Leukemia ; etiology ; genetics ; Leukemia Inhibitory Factor ; biosynthesis ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; biosynthesis ; genetics ; physiology

Triptolide( TP) is isolated from the traditional Chinese medicine Tripterygium wilfordii,which exhibits notable immuneregulative effect. Th17 cells involve in inflammatory response and Treg cells contribute to immune tolerance. They both play an important role in immune response. Previous studies have investigated that TP induced hepatic Th17/Treg imbalance. However,the effect of TP on spleen Th17/Treg cells remains unclear. Therefore,the aim of present study was to investigate the effect of TP on Th17/Treg cells in spleen. In this study,the effect of TP on the proliferation of splenic lymphocyte was detected by cytotoxicity test in vitro. After different concentrations of TP( 2. 5,5,20,40 nmol·L~(-1)) were given to splenic lymphocyte,cytokines secreted from the supernatant of splenic lymphocyte were detected by cytometric bead array,and the expression of suppressor of cytokine signaling( SOCS) mRNA was detected by qRT-PCR. Female C57 BL/6 mice were continuously observed for 24 h after treatment of 500 μg·kg-1 TP. The effects of TP on the splenic tissue structure and the percentage of Th17/Treg cells were examined. The results showed that the IC50 of TP was19. 6 nmol·L~(-1) in spleen lymphocytes. TP inhibited the secretion of IL-2 and IL-10 and induced the expression of SOCS-1/3 mRNA in spleen lymphocytes at the dosage of 2. 5 and 5 nmol·L~(-1) after 24 h in vitro. Administration of TP at dosage of 500 μg·kg-1 had no significant spleen toxicity in vivo. TP treatment increased the percentage of Th17 cells after 12 h and inhibited the proportion of Treg cells after 12 and 24 h. In conclusion,TP reduced the secretion of IL-2 and IL-10 through SOCS-1/3 signaling pathway,thereby induced the percentage of Th17 cells and inhibited the percentage of Treg cells.
Animals ; Cytokines ; metabolism ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Female ; Mice ; Mice, Inbred C57BL ; Phenanthrenes ; pharmacology ; Signal Transduction ; Spleen ; cytology ; drug effects ; Suppressor of Cytokine Signaling 1 Protein ; metabolism ; Suppressor of Cytokine Signaling 3 Protein ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

BACKGROUNDIdiopathic pulmonary fibrosis (IPF) is a progressive diffuse parenchymal disease with a poor prognosis. A variety of cytokines and chemokines are involved in its pathophysiology. The aim of this study was to evaluate the clinical features in IPF patients with the expression of suppressor of cytokine signaling 1 (SOCS-1), which acts as a negative regulator of cytokine signaling.

METHODSIPF patients (n = 20) and healthy controls (n = 16) were included in this study. The expression of SOCS-1 was analyzed in peripheral blood mononuclear cells (PBMC) of subjects using RT-PCR. Interleukin 4 (IL-4), transforming growth factor β1 (TGF-β1) and type I collagen expression were also analyzed in each individual using enzyme-linked immunosorbent assay (ELISA). The clinical characteristics of IPF patients were delineated. These results were analyzed by SPSS13.0 statistics software.

RESULTSSOCS-1 mRNA expression was significantly decreased in the PBMC of IPF patients compared with healthy controls; serum levels of IL-4 and TGF-β1 were higher in IPF patients. The patients with lower expression of SOCS-1 developed lower percentage of forced vital capacity (FVC%) and DLCO/VA. A patients' SOCS-1 mRNA level was negatively correlated with serum levels of IL-4, and negatively correlated with their high-resolution computed tomography (HRCT) scores.

CONCLUSIONSSOCS-1 mRNA can be detected in PBMC, and it is down-regulated in IPF patients. The expression of SOCS-1 is associated with the severity of IPF patients' symptoms, so it might be the predictor of disease severity. SOCS-1 might play an important role in IPF by reducing the expression of the T helper type 2 (Th2) cell-related cytokine IL-4.

Adult ; Aged ; Aged, 80 and over ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Idiopathic Pulmonary Fibrosis ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism

In order to investigate the suppressor of cytokine signaling-1 (SOCS-1) expression in peripheral blood mononuclear cells (PBMNC) of patients with acute and chronic myeloid leukemia and analyze its clinical significance, RT-PCR method was used for detecting SOCS-1 mRNA expression in PBMNC of 50 newly diagnosed patients. The result showed that positive expressions of SOCS-1 were observed in 4 of 25 patients with AML (16.00%), in 11 of 25 patients with CML (44.00%) and none in 10 normal controls. The differences between patients with AML and normal controls, and between patients with CML and normal controls were statistically significant. In CML group, 2 out of 12 cases with non-progression (chronic phase), 9 of 13 cases with progression showed the positive expression, the difference between two subgroups was statistically significant. Those CML patients with SOCS-1 mRNA expression had poor response to IFN-alpha. When they transformed into accelerated phase, SOCS-1 mRNA expression was more persistently and frequently observed, and no response to IFN-alpha was observed. Most of them had very poor prognosis. It is concluded that the SOCS-1 mRNA can be detected in the PBMNC of the patients with acute and chronic myeloid leukemia. The SOCS-1 mRNA expression in the patients with CML is higher than that in patients with AML, and it is higher in accelerated phase and blast crisis significantly. This phenomenon is highly related to the reaction of IFN-alpha and prognosis.
Humans ; Interferon-alpha ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; biosynthesis ; genetics

The aim of this study was to explore the effect of arsenic trioxide (As(2)O(3)) on the methylation status of socs-1 gene in multiple myeloma cell lines U266, RPMI8226. The cell viability was assayed by MTT method. The methylation status of socs-1 gene was detected by methylation specific PCR. The expression of socs-1 gene mRNA was determined with real-time PCR. The cell apoptosis was analyzed by flow cytometry. The results indicated that hypermethylation of CpG island of socs-1 gene was observed without expression of socs-1 in myeloma cell lines U266, RPMI8226. The expression of socs-1 gene mRNA in each myeloma cell line increased significantly after exposure to As(2)O(3) for 72 hours as compared with the cell lines of wild type (p < 0.05). And cell proliferation was significantly inhibited, both early apoptosis and later apoptosis ratios increased in dose-dependent manner. It is concluded that As(2)O(3) may induce socs-1 demethylation and up-regulate the expression of the gene. This study provides a new thought and direction for exploring possible mechanism of cell apoptosis induced by As(2)O(3) and multiple myeloma treatment by As(2)O(3).
Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Line, Tumor ; CpG Islands ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Humans ; Multiple Myeloma ; genetics ; Oxides ; pharmacology ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics

OBJECTIVETo investigate the effect of DNA methyhransferase l (DNMT1) gene silencing on methylation of suppressor of cytokine signaling (SOCS-1) in multiple myeloma RPMI 8226 cells.

METHODSRecombinant plasmid pshRNA-DNMTl was transfected into multiple myeloma RPMI 8226 cells by lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of DNMTl in RPMI 8226 cells respectively before and after transfection. Methylation-specific polymerase chain reaction was used to detect the methylation level change of SOCS-1 gene in RPMI8226 cells transfected.

RESULTSDNMTl targeted short hairpin RNA(shRNA) was successfully inserted into the plasmid vector pshRNA. RT-PCR results showed that the relative mRNA expression level of DNMTI gene in RPMI 8226 cells transfected with pshRNA was 0.176±0.004 which was significantly lower than that in cells transfected by empty vector (0.956±0.033, P<0.01). Western blot analysis showed that the relative expression level of DNMT1 protein of RPMI 8226 cells transfected by pshRNA was 0.065±0.014, which was significantly lower than that in transfected cells by empty vector(0.415±0.027) (P<0.05). These results indicated that the recombinant plasmid pshRNA could effectively knock down the expression of DNMT1 gene in RPMI 8226 cells. Methylation analysis showed that the methylation level of SOCS-1 gene was obviously reduced after transfection.

CONCLUSIONDNMT1 gene in RPMI 8226 cell can be silenced by shRNA. DNMT1 gene silencing can significantly induce SOCS-1 gene hypomethylation, which indicates that DNMT1 may play an important role in the process of SOCS-1 hypermethylation.

Cell Line, Tumor ; DNA Methylation ; Gene Silencing ; Genetic Vectors ; Humans ; Multiple Myeloma ; RNA, Messenger ; RNA, Small Interfering ; Repressor Proteins ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; Transfection

OBJECTIVETo investigate the effect of hepatitis B virus-encoded X protein (HBx) on the expression of host-encoded suppressor of cytokine signaling-1 (SOCS-1) and to explore the possibility of an underlying mechanism involving modulation of CpG island methylation in the SOCS-1 gene promoter.

METHODSThe immortalized human derived non-tumor liver cell line QSG7701 was transfected with a recombinant HBx plasmid (pcDNA-X) or an empty vector control plasmid (pcDNA3.0) and stably transfected clones were selected by G418 resistance screening. Untransfected cells served as negative controls. Expression of SOCS-1 mRNA and protein was detected by real-time quantitative PCR and western blotting. The methylation status of SOCS-1 was detected by methylation-specific PCR (MSP). The significance of intergroup differences was analyzed by one-way ANOVA or pairwise comparison with post-hoc LSD test.

RESULTSSOCS-1 mRNA level was significantly lower in the pcDNA-X/QSG7701 cells compared to that in the pcDNA3.0/QSG7701 and untransfected cells (0.3249+/-0.0536 vs. 1.0543+/-0.1937 and 1.00; F = 19.6, P = 0.042). SOCS-1 protein level was similarly lower in the pcDNA-X/QSG7701 cells (0.1496+/-0.0106 vs. 0.1984+/-0.0438 and 0.2152+/-0.0816; F = 19.4, P = 0.048). The SOCS-1 promoter region showed methylation only in the pcDNA-X/QSG7701 cells.

CONCLUSIONHBx-expressing human hepatocytes have down-regulated SOCS-1 expression, both at the mRNA and protein levels, and this effect corresponds to increased methylation in the SOCS-1 promoter region harboring CpG islands.

Cell Line ; CpG Islands ; DNA Methylation ; Humans ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; metabolism ; Trans-Activators ; genetics ; metabolism ; Transfection

Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Fetal Blood ; Humans ; Infant, Newborn ; Lipopolysaccharides ; Lymphocytes ; metabolism ; Male ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; Time Factors ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVESuppressors of cytokine signaling (SOCS) have been shown to play an important role in regulating cytokines, such as intracellular interferon (IFN) and interleukin (IL), in the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. At present, the association between SOCS and asthma is still under study. The aim of this study is to explore the relationship of SOCS-1 and SOCS-3 mRNA expression in peripheral blood mononuclear cells (PBMCs) with the intracellular IFN-'/IL-4 ratio in CD4+ T cells and specific IgE (sIgE) level in children with asthma.

METHODSBMCs were collected from 44 children with allergic asthma (4-14 years) and 30 healthy children. The intracellular IFN-'/IL-4 ratio in CD4+ T cells was measured by flow cytometry. Total RNAs were extracted from the PBMCs and SOCS-1 and SOCS-3 mRNA expression was measured by SYBR Green I quantitative RT-PCR.

RESULTSCompared with the healthy children, children with allergic asthma showed a lower level of intracellular IFN-' in peripheral blood [(15.7±2.0)% vs (19.1±2.7)%] and IFN-'/IL-4 ratio (3.4±1.5 vs 4.8±2.9) and higher SOCS-1 mRNA expression (-Ct, 11.1±1.9 vs 12.6±2.8). There was a negative relationship between SOCS-1 mRNA expression and the percentage of IFN-'-producing CD4+ T cells in peripheral blood in both asthmatic and healthy children (P<0.05). No correlation was found between SOCS-1 and SOCS-3 expression and sIgE level.

CONCLUSIONSChildren with allergic asthma have elevated levels of SOCS-1 mRNA in PBMCs, which is associated with Th2-skewed immune response.

Adolescent ; Asthma ; immunology ; Child ; Child, Preschool ; Cytokines ; genetics ; Female ; Gene Expression Regulation ; Humans ; Interferon-gamma ; genetics ; Interleukin-4 ; genetics ; Male ; RNA, Messenger ; analysis ; Signal Transduction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; Th1 Cells ; immunology ; Th2 Cells ; immunology