Suppressor Of Cytokine Signaling 3 mRNA Expression Research Articles

The importance of SOCS1 - 1478 CA/del polymorphism and expression in breast cancer: a case-control study in the north of Iran.

This project aimed to evaluate the relationship between the suppressor of cytokine signaling-1 (SOCS1) - 1478 CA > del genetic variation and breast cancer susceptibility. Moreover, we investigated the SOCS1 mRNA expression level in cancerous tissues. A total of 100 patients with breast cancer and 120 healthy individuals were selected. Genomic DNA was extracted from blood. SOCS1 genotyping and relative gene expression were performed using ARMS-PCR (Amplification-Refractory Mutation System-Polymerase Chain Reaction) and real-time PCR, respectively. In breast cancer patients, the prevalence of genotype frequencies of SOCS1 (- 1478 CA > del) CA/CA, CA/del, and del/del was 52, 31, and 17%, respectively. Among controls, the distribution of CA/CA, CA/del, and del/del was 63, 15, and 22%, respectively. The chi-square test reported that a significant difference was observed in the genotypic distribution of SOCS1 (- 1478 CA > del) polymorphism between cases and controls (χ2 = 8.08, P = 0.01). In addition, the presence of the CA/del genotype was associated with an elevated risk of breast cancer (in the codominant model: OR 2.51; 95% CI 1.27-4.96, P = 0.007 and in the over dominant model: OR 2.54; 95% CI 1.32-4.90, P = 0.005). However, there was no significant difference in allelic distributions between the groups (P > 0.05). There was no significant difference in the breast cancer risk associated with the dominant and recessive genetic models when the reference was CA/CA and CA/CA + CA/del genotype, respectively (P = 0.09 and P = 0.38). Moreover, the expression of SOCS1 decreased in cancerous tissues as compared to the adjacent non-cancerous tissues (P < 0.0001). In conclusion, a functional SOCS1 promoter polymorphism (- 1478 CA > del) may affect breast cancer susceptibility.

The Relevance of SOCS1 Methylation and Epigenetic Therapy in Diverse Cell Populations of Hepatocellular Carcinoma.

The suppressor of cytokine signaling 1 (SOCS1) is a tumor suppressor gene found to be hypermethylated in cancers. It is involved in the oncogenic transformation of cirrhotic liver tissues. Here, we investigated the clinical relevance of SOCS1 methylation and modulation upon epigenetic therapy in diverse cellular populations of hepatocellular carcinoma (HCC). HCC clinical specimens were evaluated for SOCS1 methylation and mRNA expression. The effect of 5-Azacytidine (5-AZA), a demethylation agent, was assessed in different subtypes of HCC cells. We demonstrated that the presence of SOCS1 methylation was significantly higher in HCC compared to peri-HCC and non-tumoral tissues (52% vs. 13% vs. 14%, respectively, p < 0.001). In vitro treatment with a non-toxic concentration of 5-AZA significantly reduced DNMT1 protein expression for stromal subtype lines (83%, 73%, and 79%, for HLE, HLF, and JHH6, respectively, p < 0.01) compared to cancer stem cell (CSC) lines (17% and 10%, for HepG2 and Huh7, respectively), with the strongest reduction in non-tumoral IHH cells (93%, p < 0.001). 5-AZA modulated the SOCS1 expression in different extents among the cells. It was restored in CSC HCC HepG2 and Huh7 more efficiently than sorafenib. This study indicated the relevance of SOCS1 methylation in HCC and how cellular heterogeneity influences the response to epigenetic therapy.

1003 Suppressor of cytokine signaling-1: a potential mechanistic target the immune system imbalance in preeclampsia development

Although the etiology of preeclampsia (PEC) is not completely understood, the importance of abnormal placentation in disease development is well established. This abnormal placentation is thought to be due to an immune system imbalance, characterized by an increase in proinflammatory CD4+T cells, and a decrease in regulatory T cells (Tregs). To date, the mechanism by which this imbalance develops is unknown. Notably, Suppressor of Cytokine Signaling-1 (SOCS1) is an important regulator of proinflammatory CD4+ T cells. It also plays a major role in maintaining the stability of Tregs. Moreover, SOCS1 deficiency leads to autoimmune disease development, as SOCS1 deficient mice develop lupus, and lupus patients have reduced SOCS1 expression. Given that disease development in PEC results from an imbalance toward a proinflammatory state, the objective of this study is to determine whether there are differences in SOCS1 expression between PEC patients and controls. SOCS1 mRNA expression was measured from maternal whole blood drawn during delivery admission from patients with PEC, per ACOG guidelines, and controls. All participants were women with singleton pregnancies, >37 weeks. Controls were matched for gestational age, race, and ethnicity. Exclusion criteria: patients with autoimmune disease, chronic viral infection, and/or on immunosuppressants. RNA was isolated from whole blood and analyzed using NanoDrop technology. Following cDNA synthesis, SOCS1 mRNA expression was determined by qPCR. n=5 for each group. Statistical analysis: two-tailed T test. SOCS1 expression is significantly decreased in PEC patients in comparison to controls. SOCS1 expression was decreased in this small cohort of PEC patients. Given that SOCS1 prevents uncontrolled proinflammatory cytokine expression, these preliminary results suggest that decreased SOCS1 expression in a larger cohort of PEC patients could serve as a mechanistic target for the study of the immune system imbalance in PEC development.

Effects of miR-152-Mediated Targeting of SOCS3 on Hepatic Insulin Resistance in Gestational Diabetes Mellitus Mice

BackgroundThe present study explored the effects of miR-152-mediated targeting of suppressor of cytokine signaling 3 (SOCS3) on hepatic insulin resistance (HIR) in mice with gestational diabetes mellitus (GDM). Healthy SPF C57BL/6J mice were selected to establish a GDM model. MethodsMice were divided into seven groups as follows: Normal group, Model group, NC-mimic group, miR-152 mimic group, NC-pcDNA3.0 group, pcDNA3.0-SOCS group, and miR-152 mimic + pcDNA3.0-SOCS3 group. The relationship between miR-152 and SOCS3 expression was analyzed by a dual-luciferase reporter system. Islet cell morphology and expression of miR-152 and SOCS3 mRNA and protein in the islet tissue were detected by hematoxylin and eosin (HE) staining, reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and western blot analysis. Fasting blood glucose (FBG) level was measured by a glucose meter while triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and insulin levels were determined by an automatic biochemical analyzer. Insulin resistance index (HOMA-IR) was calculated by the formula FBG × FINS/22.5. ResultsThe results showed that the levels of miR-152, SOCS3, FBG, fasting insulin (FINS), TG, and TC increased, and HDL-C content decreased in other groups as compared with those in the Normal group (all p < 0.05). Oral glucose tolerance test (OGTT), FBG, FINS, TG, TC, and HDL-C values showed an opposite trend in miR-152 mimic and pcDNA3.0-SOCS3 groups as compared with the Model group (all p < 0.05). ConclusionsmiR-152 can inhibit HIR in GDM mice by downregulating the expression of SOCS3.

Identification of a Human SOCS1 Polymorphism That Predicts Rheumatoid Arthritis Severity.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by an autoimmune response in the joints and an exacerbation of cytokine responses. A minority of patients with RA experience spontaneous remission, but most will show moderate/high disease activity, with aggressive joint damage and multiple systemic manifestations. There is thus is a great need to identify prognostic biomarkers for disease risk to improve diagnosis and prognosis, and to inform on the most appropriate therapy. Here we focused on suppressor of cytokine signaling 1 (SOCS1), a physiological negative regulator of cytokines that modulates cell activation. Using four independent cohorts of patients with arthritis, we characterized the correlation between SOCS1 mRNA levels and clinical outcome. We found a significant inverse correlation between SOCS1 mRNA expression and disease activity throughout the follow-up of patients with RA. Lower baseline SOCS1 levels were associated with poorer disease control in response to methotrexate and other conventional synthetic disease-modifying anti-rheumatic drugs in early arthritis, and to rituximab in established (active) RA. Moreover, we identified several single nucleotide polymorphisms in the SOCS1 gene that correlated with SOCS1 mRNA expression, and that might identify those patients with early arthritis that fulfill RA classification criteria. One of them, rs4780355, is in linkage disequilibrium with a microsatellite (TTTTC)3−5, mapped 0.9 kb downstream of the SNP, and correlated with reduced SOCS1 expression in vitro. Overall, our data support the association between SOCS1 expression and disease progression, disease severity and response to treatment in RA. These observations underlie the relevance of SOCS1 mRNA levels for stratifying patients prognostically and guiding therapeutic decisions.

241-LB: Positive Regulation of JAK/STAT at Signaling with SOCS3-Derived Cell-Permeable Binding Domain to ObR Induces Anti-obesity Effect

Aim: Leptin, a hormone which regulates food intake and energy expenditure, is excessively expressed in obese patients. Leptin signaling is regulated by a negative feedback regulator, suppressor of cytokine signaling 3 (SOCS3) that competitively binds to the leptin receptor (ObR) and promotes leptin resistance. We hypothesized that an exogenously delivered SOCS3 binding domain (SH2) would compete with SOCS3 for binding to p-Tyr985ObR, and thereby maintain leptin-initiated JAK/STAT signaling. Method: Cell-permeable, dominant-interfering ΔSOCS3 protein consists of the SOCS3 SH2 domain, a sequence optimized-advanced macromolecule transduction domain to deliver the protein into cells and tissues to facilitate soluble purification, biologically active proteins. Intracellular delivery of ΔSOCS3 was tested for the ability to enhance leptin signaling and sensitivity in diet-induced obese (DIO) mice. Result: The cell-permeable ΔSOCS3 was delivered efficiently into cell types and tissues examined including the hypothalamus; thus, the protein appeared to cross the blood-brain barrier. CP-ΔSOCS3 competitively bound p-Tyr985ObR and blocked negative regulation by endogenous SOCS3 as assessed by increased levels of phospho-STAT3. DIO mice treated with CP-ΔSOCS3 showed 12.1% body weight loss in 14 days. In addition, fat volume (53.5%), food intake (29.2%), serum leptin (93.1%) and expression of SOCS3 mRNA were also decreased in CP-ΔSOCS3-treated compared to mice treated with diluent. In addition, treatment group showed a significant increase in STAT3 phosphorylation and pro-opiomelanocortin in the hypothalamus. Conclusion: CP-ΔSOCS3 appeared to competitively suppress feedback inhibition of JAK/STAT signaling by endogenous SOCS3 thereby enhancing leptin sensitivity. This establishes the importance of SOCS3 in the biology of leptin signaling and resistance and suggests CP-ΔSOCS3 might be used as a mechanism-specific weight-loss therapy. Disclosure K. Lee: None. J. Jeon: None. S. Kim: None. H. Kang: None. J. Kwak: None. Y. Choi: None. D. Jo: None.

LncGBP9/miR-34a axis drives macrophages toward a phenotype conducive for spinal cord injury repair via STAT1/STAT6 and SOCS3

BackgroundAcute spinal cord injury (SCI) could cause mainly two types of pathological sequelae, the primary mechanical injury, and the secondary injury. The macrophage in SCI are skewed toward the M1 phenotype that might cause the failure to post-SCI repair.MethodsSCI model was established in Balb/c mice, and the changes in macrophage phenotypes after SCI were monitored. Bioinformatic analyses were performed to select factors that might regulate macrophage polarization after SCI. Mouse bone marrow-derived macrophages (BMDMs) were isolated, identified, and induced for M1 or M2 polarization; the effects of lncRNA guanylate binding protein-9 (lncGBP9) and suppressor of cytokine signaling 3 (SOCS3) on macrophages polarization were examined in vitro and in vivo. The predicted miR-34a binding to lncGBP9 and SOCS3 was validated; the dynamic effects of lncGBP9 and miR-34a on SOCS3, signal transducer and activator of transcription 1 (STAT1)/STAT6 signaling, and macrophage polarization were examined. Finally, we investigated whether STAT6 could bind the miR-34a promoter to activate its transcription.ResultsIn SCI Balb/c mice, macrophage skewing toward M1 phenotypes was observed after SCI. In M1 macrophages, lncGBP9 silencing significantly decreased p-STAT1 and SOCS3 expression and protein levels, as well as the production of Interleukin (IL)-6 and IL-12; in M2 macrophages, lncGBP9 overexpression increased SOCS3 mRNA expression and protein levels while suppressed p-STAT6 levels and the production of IL-10 and transforming growth factor-beta 1 (TGF-β1), indicating that lncGBP9 overexpression promotes the M1 polarization of macrophages. In lncGBP9-silenced SCI mice, the M2 polarization was promoted on day 28 after the operation, further indicating that lncGBP9 silencing revised the predominance of M1 phenotype at the late stage of secondary injury after SCI, therefore improving the repair after SCI. IncGBP9 competed with SOCS3 for miR-34a binding to counteract miR-34a-mediated suppression on SOCS3 and then modulated STAT1/STAT6 signaling and the polarization of macrophages. STAT6 bound the promoter of miR-34a to activate its transcription.ConclusionsIn macrophages, lncGBP9 sponges miR-34a to rescue SOCS3 expression, therefore modulating macrophage polarization through STAT1/STAT6 signaling. STAT6 bound the promoter of miR-34a to activate its transcription, thus forming two different regulatory loops to modulate the phenotype of macrophages after SCI.

Visceral Adipose Tissue Inflammatory Factors (TNF-Alpha, SOCS3) in Gestational Diabetes (GDM): Epigenetics as a Clue in GDM Pathophysiology

Gestational diabetes (GDM) is among the most challenging diseases in westernized countries, affecting mother and child, immediately and in later life. Obesity is a major risk factor for GDM. However, the impact visceral obesity and related epigenetics play for GDM etiopathogenesis have hardly been considered so far. Our recent findings within the prospective ‘EaCH’ cohort study of women with GDM or normal glucose tolerance (NGT), showed the role, critical factors of insulin resistance (i.e., adiponectin, insulin receptor) may have for GDM pathophysiology with epigenetically modified expression in subcutaneous (SAT) and visceral (VAT) adipose tissues. Here we investigated the expression and promoter methylation of key inflammatory candidates, tumor necrosis factor-alpha (TNF-α) and suppressor of cytokine signaling 3 (SOCS3) in maternal adipose tissues collected during caesarian section (GDM, n = 19; NGT, n = 22). The mRNA expression of TNF-α and SOCS3 was significantly increased in VAT, but not in SAT, of GDM patients vs. NGT, accompanied by specific alterations of respective promoter methylation patterns. In conclusion, we propose a critical role of VAT and visceral obesity for the pathogenesis of GDM, with epigenetic alterations of the expression of inflammatory factors as a potential factor.

Protective effect of resveratrol on obesity-related osteoarthritis via alleviating JAK2/STAT3 signaling pathway is independent of SOCS3

Resveratrol (RES) has a protective effect on osteoarthritis (OA), nevertheless, the underlying mechanisms of RES towards obesity-related OA are still unclear. This study is aimed to determine whether leptin resistant mechanism presents in articular cartilage of obesity-related OA and whether the protective effect of RES is involved in the regulation of leptin signal by affecting suppressor of cytokine signaling 3 (SOCS3). Male C57BL/6 J mice were fed with standard chow diet, high fat diet (HFD) or high fat diet with RES (45 mg/kg.bw) for 22 weeks. Knee joints of mice were evaluated by histological and immunohistochemistry analysis. Serum level of leptin was measured by ELISA. The leptin, leptin receptor (OB-Rb), SOCS3 mRNA expression and JAK2, STAT3, OB-Rb and SOCS3 protein expression in cartilage were determined by real-time RT-PCR and western blot. In addition, SW1353 cells were pretreated with or without AG490, and stimulated with leptin in the presence or absence of RES to detect JAK2, STAT3, matrix metalloproteinase-13 (MMP-13) and SOCS3 expression. We found that HFD could induce obesity-related OA and RES prevented its progression. Serum leptin and mRNA expression in cartilage was up-regulated by HFD, while RES ameliorated the elevation. Besides, RES significantly inhibited the JAK2/STAT3 signaling pathway in cartilage, as well as SOCS3. In in vitro study, RES exhibited the same effect in SW1353 cells which stimulated with leptin. In conclusion, no significant leptin resistance existed in cartilage of obesity-related OA and the inhibitory effect of RES on obesity-related OA via alleviating JAK2/STAT3 signaling pathway is independent of SOCS3.

Relationship between the suppressor of cytokine signaling 3 expression and antiviral efficacy of nucleos(t)ide and interferon alpha therapy for chronic hepatitis B

Objective: To investigate the molecular mechanism of poor response of nucleoside and interferon therapy in some patients with chronic hepatitis B (CHB) and the negative regulatory factor of suppressor of cytokine signaling 3 (SOCS3) expression in the interferon-signaling pathway. Also, study the clinical relationship between SOCS3 and antiviral efficacy of nucleoside and interferon. Methods: Peripheral blood and matched liver tissue samples from 54 CHB patients who participated in the OSST study were selected. HBsAg was measured at different time points (baseline and weeks 12, 24, 36, and 48) to observe the antiviral efficacy. Meanwhile, quantitative real-time PCR, and immunohistochemistry were used to detect the expression levels of SOCS3 mRNA in peripheral blood mononuclear cells (PBMCs) and matched liver tissues (baseline and 48 weeks). At the end of the 48-week treatment, patients with HBsAg negative or HBeAg seroconversion were defined as response group, and vice versa. Paired t-tests were used to compare normal distribution variables and the Mann-Whitney U test was used to compare the median differences between groups of non-normally distributed variables. Results: After 48 weeks of treatment, serum HBsAg levels in the Peg-IFN group continued to decline (average decrease of 1.14 log(10) IU / ml at week 48; P = 0.001 compared with baseline), while the entecavir group remained almost unchanged during treatment (average decrease was 0.05 log(10) IU / ml at week 48; compared with baseline P = 0.12). The expression of SOCS3 mRNA (Messenger RNA, mRNA) in peripheral blood and liver tissues of non-responder group was significantly higher than the response group in the course of Peg-IFNα2a treatment. The immunohistochemical results of liver tissue showed that the expression of SOCS3 in the non-responder group was significantly higher than that in the response group at baseline (P = 0.027). After 48 weeks of treatment with Peg-IFNα2a, the expression of SOCS3 in the non-responder group was significantly higher than that in the baseline and response groups (P = 0.003, P = 0.012, respectively). Conclusion: The expression of SOCS3 in peripheral blood mononuclear cells and liver tissues of non-responding CHB patients was significantly higher than that of responding CHB patients during interferon and nucleoside antiviral therapy. We speculated that SOCS3 might affect the antiviral efficacy through negative regulation of JAK-STAT signaling pathway, and partly expose the mechanism of interferon resistance.

Role of hypomethylation of suppressor of cytokine signaling in T helper 17 cell/regulatory T cell imbalance in children with Henoch-Schönlein purpura

To investigate the association between suppressor of cytokine signaling (SOCS) hypomethylation and T helper 17 (Th17) cell/regulatory T (Treg) cell imbalance in children with Henoch-Schönlein purpura (HSP) and the immune pathogenesis of HSP. A total of 32 children in the acute stage of HSP who were hospitalized from May 2014 to January 2015 were enrolled as subjects, and 28 children who underwent physical examination were enrolled as normal control group. ELISA was used to measure the plasma level of interleukin-6 (IL-6). Flow cytometry was used to measure the percentages of CD4+ IL-17A+ T cells (Th17 cells) and CD4+CD25+ Treg cells (Treg cells) in peripheral blood and mean fluorescence intensity (MFI) for phosphorylated-STAT3 (pSTAT3) protein in CD4+ T cells. Quantitative real-time PCR was used to measure the mRNA expression of suppressor of cytokine signaling-1 (SOCS1) and suppressor of cytokine signaling-3 (SOCS3) in CD4+ T cells. High-resolution melting (HRM) was used to evaluate the methylation level of the CpG islands in SOCS1 exon 2 and the CpG islands of the potential bind sites for STAT3 in the 5'-untranslated region (5'-UTR) of SOCS3 in peripheral blood mononucleated cells. Compared with the normal control group, the HSP group had significant increases in plasma IL-6 concentration and MFI for pSTAT3 in CD4+ T cells, as well as a significant increase in the percentage of Th17 cells and a significant reduction in the percentage of Treg cells (P<0.05). The HSP group had significantly higher mRNA expression of SOCS1 and SOCS3 in peripheral blood mononucleated cells than the normal control group (P<0.05). In the HSP group, the mRNA expression of SOCS1 and SOCS3 was negatively correlated with Th17/Treg ratio (P<0.05). The HSP group had hypomethylation of the CpG islands in SOCS1 exon 2 and the potential binding site for STAT3 in SOCS3 5'-UTR, while the normal control group had complete demethylation. Low relative expression of SOCS1 and SOCS3 caused by hypomethylation may be a factor for Th17/Treg imbalance in children with HSP.

Upregulation of Suppressor of Cytokine Signaling 3 in Microglia by Cinnamic Acid.

Neuroinflammation plays an important role in the pathogenesis of various neurodegenerative diseases including Alzheimer's disease (AD). Suppressor of cytokine signaling 3 (SOCS3) is an anti-inflammatory molecule that suppresses cytokine signaling and inflammatory gene expression in different cells including microglia. The pathways through which SOCS3 could be upregulated are poorly described. Cinnamic acid is a metabolite of cinnamon, a natural compound that is being widely used all over the world as a spice or flavoring agent. Here, we examined if cinnamic acid could upregulate SOCS3 in microglia. Microglia and astroglia isolated from mouse brain as well as BV-2 microglial cells were treated with cinnamic acid followed by monitoring the level of SOCS3 and different proinflammatory molecules by RT-PCR and real-time PCR. To nail down the mechanism, we also performed ChIP analysis to monitore the recruitment of cAMP response element binding (CREB) to the socs3 gene promoter and carried out siRNA knockdown of CREB. Cinnamic acid upregulated the expression of SOCS3 mRNA and protein in mouse BV-2 microglial cells in dose- and time-dependent manner. Accordingly, cinnamic acid also increased the level of SOCS3 and suppressed the expression of inducible nitric oxide synthase and proinflammatory cytokines (TNFα, IL-1β and IL-6) in LPSstimulated BV-2 microglial cells. Similar to BV-2 microglial cells, cinnamic acid also increased the expression of SOCS3 in primary mouse microglia and astrocytes. We have seen that cAMP response element is present in the promoter of socs3 gene, that cinnamic acid induces the activation of CREB, that siRNA knockdown of CREB abrogates cinnamic acid-mediated upregulation of SOCS3, and that cinnamic acid treatment leads to the recruitment of CREB to the socs3 gene. These studies suggest that cinnamic acid upregulates the expression of SOCS3 in glial cells via CREB pathway, which may be of importance in neuroinflammatory and neurodegenerative disorders.

Interleukin-6 induces drug resistance in renal cell carcinoma.

Metastatic renal cell carcinoma (mRCC) is a tumor entity with poor prognosis due to limited therapy options. Tyrosine kinase inhibitors (TKIs), the novel targeted agents have been used for the treatment of mRCC and have shown efficacy. Interferon (IFN)-α is also one of the most frequently used agents in immunotherapy. However, drug resistance needs to be overcome to achieve a sufficiently positive effect. Interleukin-6 (IL-6), which induce suppressor of cytokine signaling-3 (SOCS3) expression, is one of the factors associated with poor prognosis of patients with renal cell carcinoma (RCC). To analyze the influence of IL-6 in drug resistance of RCC, anti-IL-6 receptor antibody was used in combination with IFN or TKIs. The SOCS3 mRNA expression level was significantly increased by IFN-α stimulation in 786-O RCC cells which were resistant to IFN, but not in ACHN cells that were sensitive to IFN. The overexpression of SOCS3 by gene transfection in ACHN significantly inhibited the growth-inhibitory effect of IFN-α. An in vivo study demonstrated that co-administration of SOCS3-targeted siRNA promoted INF-α-induced cell death and growth suppression in 786-O cell xenograft. SOCS3 could be a key component in the resistance to interferon treatment of renal cell carcinoma. Because SOCS3 is rapidly up-regulated by IL-6 and a negative regulator of cytokine signaling, IL-6 expression on RCC cells was also analyzed and the 786-O cells showed the high level of IL-6 mRNA expression under the condition of interferon stimulation. IL-6R antibody, tocilizumab, significantly suppressed cell proliferation in 786-O cells by interferon stimulation accompanied with phosphorylation of STAT1 and inhibited SOCS3 expression. The in vivo effects of combination therapy with tocilizumab and interferon showed significant suppression of 786-O tumor growth in a xenograft model. We also hypothesized that TKI resistance and IL-6 secretion are causally connected. And we found that 786-O RCC cells secrete high IL-6 levels after low dose stimulation with the TKIs sorafenib, sunitinib and pazopanib, inducing activation of AKT-mTOR pathway, NFκB, HIF-2α and VEGF expression. Tocilizumab neutralizes the AKT-mTOR pathway activation and results in reduced proliferation. A combination therapy with tocilizumab and TKI suppresses 786-O tumor growth and inhibits angiogenesis in vivo more efficient than TKI alone. Our findings suggest that IL-6 could induce drug resistance on RCC, and combination therapy of IL-6R inhibitors and IFN/TKIs may represent a novel therapeutic approach for RCC treatment.

Effect of transforming growth factor-β1 on HBV replication and antigen synthesis in HepG2.2.15 cells with steatosis

Objective: To investigate the effect of transforming growth factor-β1 (TGF-β1) on HBV replication and protein expression in HepG2.2.15 cells with steatosis, as well as the association of TGF-β1 with suppressor of cytokine signaling-3 (SOCS-3) mRNA and sterol regulatory element-binding protein-1c (SREBP-1c) mRNA during the steatosis of HepG2.2.15 cells. Methods: The cells were divided into HepG2/HepG2.2.15 cell control groups (C1/C2 groups) and HepG2/HepG2.2.15 cell steatosis groups (F1/F2 groups). 5 ng/ml TGF-β1 was added to the two cell systems for intervention to establish TGF-β1 intervention groups (T1/T2 groups) and steatosis+TGF-β1 intervention groups (TF1/TF2 groups). A time-resolved fluorescence analyzer was used to measure HBsAg and HBeAg, and quantitative real-time PCR was used to measure HBV DNA, SOCS-3 mRNA, and SREBP-1 mRNA. A one-way analysis of variance and a factorial analysis were used for the statistical analysis of data. Results: TGF-β1 significantly reduced the level of HBeAg in C2 group (P = 0.034) and the levels of HBsAg (P < 0.001) and HBeAg (P = 0.004) in F2 group. There was an interaction between steatosis and TGF-β1 in inhibiting HBsAg. In addition, TGF-β1 significantly reduced the mRNA expression of SOCS-3 in C1, F1, C2, and F2 groups (P < 0.05) and significantly increased the mRNA expression of SREBP-1c in C1, F1, C2, and F2 groups (P < 0.05), suggesting that there was an interaction between steatosis and TGF-β1 in downregulating the mRNA expression of SOCS-3 and upregulating the mRNA expression of SREBP-1c. Conclusion: TGF-β1 does not affect HBV duplication in HepG2.2.15 cells and can inhibit the expression of HBsAg and HBeAg. TGF-β1 can downregulate the mRNA expression of SOCS-3 and upregulate the mRNA expression of SREBP-1c.

SOCS3 genetic variants and promoter hypermethylation in patients with chronic hepatitis B.

The clinical manifestations of hepatitis B viral infection (HBV) include chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The contribution of negative regulator suppressor of cytokine signaling-3 (SOCS3) promoter variants in HBV disease and SOCS3 hypermethylation in tumor tissues were investigated The SOCS3 promoter region was screened for polymorphisms in 878 HBV patients and in 272 healthy individuals. SOCS3 promoter methylation was examined by bisulfite sequencing. SOCS3 mRNA expression was quantified in 37 tumor and adjacent non-tumor liver tissue specimens. The minor allele rs12953258A was associated with increased susceptibility to HBV infection (OR=1.3, 95%CI=1.1-1.6, adjusted P=0.03). The minor allele rs111033850C and rs12953258A were observed in increased frequencies in HCC and LC patients compared to CHB patients (HCC: OR=1.7, 95%CI=1.1-2.9, adjusted P=0.046; LC: OR=1.4, 95%CI=1.1-1.9, adjusted P=0.017, respectively). HBV patients with rs111033850CC major genotype had decreased viral load (P=0.034), whereas the rs12953258AA major genotype contributed towards increased viral load (P=0.029). Tumor tissues revealed increased hypermethylation compared to adjacent non-tumor tissues (OR=5.4; 95%CI= 1.9-17.1; P=0.001). Increased SOCS3 expression was observed in HBV infested tumor tissues than non-HBV related tumor tissues (P=0.0048). SOCS3 promoter hypermethylation was associated with relatively low mRNA expression in tumor tissues (P=0.0023). In conclusion, SOCS3 promoter variants are associated with HBV susceptibility and SOCS3 hypermethylation stimulates HCC development.

Upregulation of SOCS3 in lung CD4+ T cells in a mouse model of chronic PA lung infection and suppression of Th17‑mediated neutrophil recruitment in exogenous SOCS3 transfer in vitro.

Neutrophilic airway inflammation in chronic lung infections caused by Pseudomonas aeruginosa (PA) is associated with T helper (Th)17 responses. Suppressor of cytokine signaling 3 (SOCS3) is the major negative modulator of Th17 function through the suppression of signal transducer and activator of transcription (STAT)3 activation. The aim of the present study was to investigate the expression of SOCS3 in lung CD4+ T cells in a mouse model of chronic PA lung infection and the effect of exogenous SOCS3 on Th17-mediated neutrophil recruitment in vitro. A mouse model of chronic PA lung infection was established and the activation of STAT3 and Th17 response in lung tissues and lung CD4+ T cells was assessed. The protein and mRNA expression of SOCS3 in lung CD4+ T cells was analyzed by western blotting and reverse transcription-quantitative polymerase chain reaction. The authors constructed a recombinant lentivirus carrying the SOCS3 gene and transferred it into lung CD4+ T cells isolated from a mouse model. These transfected cells were stimulated with interleukin (IL)-23 in vitro and the protein level of p-STAT3 and retinoid-related orphan receptor (ROR)γt was determined by western blotting. The expression of IL-17A+ cells was analyzed by flow cytometry and the level of IL-17A in cell culture supernatant was measured by ELISA. The mouse lung epithelial cell line, MLE-12, was cocultured with lung CD4+ T cells that overexpressed the SOCS3 gene and the culture supernatant was harvested and used for a chemotaxis assay. Compared with control mice, mice with chronic PA lung infection had significantly higher level of p-STAT3 and Th17 response in both lung tissues and lung CD4+ T cells. The protein and mRNA level of SOCS3 in lung CD4+ T cells increased as the chronic PA lung infection developed. Exogenous SOCS3 gene transfer in PA-infected lung CD4+ T cells decreased p-STAT3 and RORγt expression and suppressed the level of IL-17A+ cells in vitro. MLE-12 cells cocultured with SOCS3-overexpressing lung CD4+ T cells expressed a significantly lower level of neutrophil chemoattractants chemokine (C-X-C motif) ligand (CXCL) 1 and CXCL5, and recruited significantly smaller numbers of migrating neutrophils than those cocultured with control cells. SOCS3 was upregulated in lung CD4+ T cells following the activation of STAT3/Th17 axis in a mouse model of chronic PA lung infection. Exogenous SOCS3 transfer in PA-infected lung CD4+ T cells suppresses Th17-mediated neutrophil recruitment in vitro.

A conserved inhibitory role of suppressor of cytokine signaling 1 (SOCS1) in salmon antiviral immunity

The SOCS proteins are regulators of JAK/STAT signaling. A number of viral infections has been associated with SOCS upregulation. Here we report that SOCS1 mRNA expression is up-regulated in salmon alphavirus (SAV3) infected TO cells, while infectious pancreatic necrosis virus infection has a negligible effect. SAV3 infected salmon showed increased SOCS1 mRNA levels in heart correlating with increased viral transcripts. Together, the in vitro and in vivo data suggests that SAV3-induced SOCS1 expression may affect the outcome of the virus infection. Using CHSE-214 cells overexpressing SOCS1 we revealed increased SAV3 replication compared to control, suggesting that SOCS1 suppress the antiviral capacity of the cells. In IFNa1-treated cells, the suppression of viral replication was partially rescued by SOCS1 overexpression. The increased viral replication in SOCS1 transgene cells was likely a result of impaired IFN-signaling and the reduced expression of interferon-stimulated genes in the transgene cells underscores this.

SOCS3 mRNA expression and polymorphisms as pretreatment predictor of response to HCV genotype 3a IFN-based treatment.

AimSuppressor of Cytokine Signaling 3 (SOCS3) gene belongs to SOCS family as one of the negative regulators of cytokine signaling and IFN response that function via the JAK-STAT pathway in antiviral response. SOCS3 expression and genetic polymorphism influences the pathogenesis and outcome of antiviral treatment in hepatitis C virus (HCV) infected patients. This study was designed for analysis of SOCS3 gene expression and polymorphism in Pakistani HCV patients.MethodsThis descriptive study was conducted on 250 diagnosed HCV genotype 3a infected subjects. The study population was divided into two major groups on the basis of therapeutic response i.e. sustained virological response (SVR) and non-responders/relapsers (NR). SOCS3 gene mRNA expression analysis was done by using Real time PCR technique, whereas ARMS PCR technique was used for analysis of SOCS3 gene polymorphisms i.e. 8464 A/C (rs12952093), −4874 A/G (rs4969170) and −1383 A/G, (rs4969168).ResultsGene expression analysis of SOCS3 showed that there was statistically significant increase of 2.275-fold and 3.72-fold in relative gene expression for SVR and NR as compared to normal healthy samples (p < 0.001). The distribution of rs4969168, rs4969170 and rs12952093 genotype frequencies between SVR versus NR group were not statistically significant, only the allelic frequency of rs4969170 was statistically significant (p ≤ 0.0001) with therapeutic response.ConclusionThe gene expression analysis of SOCS3 showed a clear difference in mRNA expression of SOCS3 as a possible indicator of therapeutic response rather than polymorphism of SOCS3 gene in our studied population.

The regulative effcets of A2a adenosine receptor on expression of SOCS-3 in rats of hypoxic pulmonary hypertension

To study the regulative effects and mechanism of A2aAR on expression of suppressor of cytokinesignaling-3(SOCS-3) in hypoxic pulmonary hypertension rats. Sprague-Daeley rats were randomly divided into 3 groups: a normal control group, a hypoxia group, and a hypoxia with selective agonists of A2aAR group. Animals in the hypoxia groups were housed in a chamber with 8%- 11% O2 and 1%-3% CO2 for 8 hours (8: 00 AM to 4: 00 PM) daily for 28 days. They were treated intraperitoneally with either 4 ml/kg weight of normal saline or 0.2 mg/kg weight of CGS-21680 30 minutes before exposure to hypoxia. Four weeks later, mean pulmonary artery pressure (mPAP), mean carotid arterial pressure (mCAP) and right ventricular rate [RV/(LV+ S)] were measured. The expression of A2aAR and SOCS-3 in pulmonary arterioles was measured by immunohistochemistry. The expression of A2aAR mRNA and SOCS-3 mRNA in lung tissues were measured by real time RT-PCR. The expression of A2aAR protein and SOCS-3 protein in lung tissues were measured by Western blot. The mPAP in the hypoxia group was [(20.9±3.9)mmHg, 1 mmHg=0.133 kPa], significantly higher than the normal control group [(12.6±6.6)mmHg](P<0.01). The mPAP in CGS-21680 group was [ (14.8±3.8)mmHg], significantly lower than the hypoxia group(P<0.01). RV/(LV+ S) in the hypoxia group was [(35.2±2.0)%] , significantly higher than the normal control group [(29.6±2.7)%] (P<0.01). RV/(LV+ S) in the CGS-21680 group was [(28.3±8.8)%], significantly lower than the hypoxia group(P<0.01). WA/TA in the hypoxia group was (73±5, P<0.01), significantly higher than the normal control group. WA/TA in CGS21680 group was (54±3, P<0.01), significantly lower than the hypoxia group. A2aAR and SOCS-3 expressions on pulmonary arterioles in the hypoxia group were (0.134±0.034) and (0.119±0.011), both significantly higher than the normal group(P<0.01); and CGS-21680 treatment further increased their expressions. The mRNA expression of both molecules showed a 1.5-fold increase after 28-day hypoxia exposure. A2aAR activation by CGS-21680 treatment in hypoxia-exposed rats further increased the expression levels of A2aAR and SOCS-3 to about 2-fold higher than the normal controls. Furthermore, protein levels of A2aAR and SOCS-3 in the lung tissue were determined using Western blot. A similar increase was observed in hypoxia-induced pulmonary hypertension, and CGS-21680 treatment group showed the highest levels of these 2 proteins. A2aAR activation prevents hypoxia-induced pulmonary hypertension, and its mechanisms are related to the activation of A2aAR SOCS-3 signaling pathway.

117. Development of Novel Cancer Therapy Combination of Ad-SOCS Gene Therapy and LAK Cell Immunotherapy for the Treatment of Prostate Cancer

Background Suppressor of cytokine signaling 3 (SOCS3) is a promising molecule for cancer gene therapy. SOCS3 suppresses tumor growth through inhibition of mutiple signaling pathways including Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase (ERK), and focal adhesion kinase (FAK). The constitutive activation of JAK/STAT3 pathway is known to be involved in oncogenesis of various types of cancers including prostate cancer. We investigated the anti-tumor activity of Ad-SOCS, which is the recombinant adenovirus vectors carrying the SOCS3 gene, against prostate cancer cell line in vitro. Furthermore, to improve the antitumor effect against prostate cancer, we combined LAK (Lymphokine activated killer) cell immunotherapy with Ad-SOCS gene therapy. Materials and Methods LNCaP, human prostate cancer cell line was used in this study. The cytotoxicity of Ad-SOCS against LNCaP was measured by XTT assay. LAK cells were generated from PBMCs isolated from fresh blood by Ficoll-paque (GE Healthcare, Munich, Germany) density gradient centrifugation. PBMC were incubated in medium with interleukin (IL)-2 and anti CD3 antibody (OKT3) to generate LAK cells. The cytotoxicity of LAK cells against LNCaP cells infected with Ad-SOCS was investigated by the in vitro cytotoxicity assay. Expression level of SOCS3, STAT3 and ULBPs were measured by real-time PCR. Results Ad-SOCS showed the significantly higher cell growth inhibitory effect compared to Ad-LacZ and PBS controls in LNCaP cells. In real-time PCR, Ad-SOCS significantly increased the level of SOCS3 mRNA expression. Sequentially, the expression of STAT3 mRNA was decreased by Ad-SOCS infection. Upregulation of SOCS3 mRNA and downregulation of STAT3 mRNA were not observed in both Ad-LacZ and PBS infected LNCaP cells. In cytotoxicity assay, LAK cells exhibited the significantly higher cytotoxicity against LNCaP cells infected with Ad-SOCS at all effector: target cell ratios, 1:1,5:1, 10:1, compared with that of non-infected or Ad-LacZ infected cells. The mRNA expression levels of ULBP 1-5, which are NKG2D ligands to stimulate LAK cell activities, were measured and the mRNA expressions of ULBP 1, 3 and 4, in LNCaP infected with Ad-SOCS were significantly increased compared to the treatments. Conclusion In the present study, we demonstrated that Ad-SOCS could inhibit the cell growth of LNCaP cells and increased the sensitivity of LAK cell in vitro. These findings suggested that the Ad-SOCS and LAK cell combination therapy warranted the further development of novel therapeutic approach for advanced prostate cancer.