Suppressor Of Cytokine Signaling 3 Protein Expression Research Articles

Jianpi Qutan Fang induces anti-atherosclerosis and ameliorates endothelial cell injury in high-fat diet ratsan anti-inflammatory and inhibiting Janus kinase/signal transducer and activator of transcription signaling pathway.

To investiage the possible mechanism underlying the effect of the Jianpi Qutan Fang (, JPQT) on Atherosclerosis (AS) which is the main pathological process of most cardiovascular diseases that affect millions of adults worldwide. In the present study, rats were fed with a high-fat-diet (HFD) with vitamin D3 for 16 weeks and were orally administered atorvastatin treatment and different doses of JPQT. Histopathological changes and ultrastructural changes in the aorta were evaluated through hematoxylin-eosin staining and transmission electron microscopy (TEM), respectively. Suppressor of cytokine signaling 1 (SOCS1)/Janus kinase 1 (JAK1)/ signal transducer and activator of transcription 1 (STAT1) signaling pathways were detected through Western blotting. JPQT treatment decreased the lipid levels of triglyceride, low-density lipoprotein, and cholesterol, the inflammatory cytokine levels of interleukin 1 beta (IL-1β), IL-6 and IL-8 in rat serum, but increased high-density lipoprotein and IL-10 serum levels. JPQT treatment ameliorated pathological changes in the aorta of AS model rats. Moreover, JPQT upregulated SOCS1 protein expression and down-regulated phosphorylated protein expression levels of p-JAK1 and p-STAT1. These results suggest that JPQT induces anti-atherosclerosis effects through anti-inflammatory and inhibiting JAK/STAT signaling pathways in HFD fed rats.

SOCS3 protein expression predicts the responses of advanced non-small cell lung cancer patients to platinum-based chemotherapy.

This study sought to assess the relationship between suppressor of cytokine signaling 3 (SOCS3) expression, SOCS3 promoter methylation status, and platinum-based chemotherapy responses in advanced non-small cell lung cancer (NSCLC) patients. A total of 400 advanced NSCLC patients with inoperable disease were enrolled in this study. All the patients underwent platinum-based chemotherapy treatment, and the clinical and prognostic outcomes of these patients were analyzed. The SOCS3 protein expression and SOCS3 promoter methylation status of the tumor tissues in these patients were also tested by immunohistochemistry and polymerase chain reaction (PCR), respectively. In addition, we knocked down SOCS3 expression via small-interfering RNA (siRNA) in the lung cancer cell lines and conducted in vitro analyses to examine cell viability and apoptosis. Patients with higher expression levels of SOCS3 were found to have a lower average tumor stage, higher average tumor differentiation, and higher rates of positive chemotherapy responses than those with lower expression levels of SOCS3. SOCS3 promoter methylation was also found to be correlated with chemotherapy responses in these patients. In the prognostic analyses, only SOCS3 expression, but not SOCS3 promoter methylation, was found to be predictive of outcomes in advanced NSCLC patients. We also found that the pro-apoptotic effects of SOCS3 were mediated by the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathways in the lung cancer cells. Currently, there is a lack of reliable biomarkers for predicting the responses of NSCLC patients to chemotherapy. Our results may aid in clinical evaluations of NSCLC patients.

Involvement of the IL-6 Signaling Pathway in the Anti-Anhedonic Effect of the Antidepressant Agomelatine in the Chronic Mild Stress Model of Depression.

Neuroinflammation has emerged as an important factor in the molecular underpinnings of major depressive disorder (MDD) pathophysiology and in the mechanism of action of antidepressants. Among the inflammatory mediators dysregulated in depressed patients, interleukin (IL)-6 has recently been proposed to play a crucial role. IL-6 activates a signaling pathway comprising the JAK/STAT proteins and characterized by a specific negative feedback loop exerted by the cytoplasmic protein suppressor of cytokine signalling-3 (SOCS3). On these bases, here, we explored the potential involvement of IL-6 signaling in the ability of the antidepressant drug agomelatine to normalize the anhedonic-like phenotype induced in the rat by chronic stress exposure. To this aim, adult male Wistar rats were subjected to the chronic mild stress (CMS) paradigm and chronically treated with vehicle or agomelatine. The behavioral evaluation was assessed by the sucrose consumption test, whereas molecular analyses were performed in the prefrontal cortex. We found that CMS was able to stimulate IL-6 production and signaling, including SOCS3 gene and protein expression, but the SOCS3-mediated feedback-loop inhibition failed to suppress the IL-6 cascade in stressed animals. Conversely, agomelatine treatment normalized the stress-induced decrease in sucrose consumption and restored the negative modulation of the IL-6 signaling via SOCS3 expression and activity. Our results provide additional information about the pleiotropic mechanisms that contribute to agomelatine’s therapeutic effects.

Effects of suppressor of cytokine signaling 3 (SOCS3) on the development of colon cancer via regulation of HIF-1α

Purpose: To investigate the influence of suppressor of cytokine signaling 3 (SOCS3) on rats with colon cancer (CC).
 Methods: Sprague-Dawley (SD) rats were randomly divided into CC group and control group. CC models were constructed. The expression of SOCS3 in CC tissues was determined by quantitative real time-polymerase chain reaction (qRT-PCR). Hematoxylin-eosin staining (H&E) was used to examine colon tissue morphology, while immunohistochemistry (IHC) staining assay was performed to determine the expression of SOCS3 protein in colon tissues. The content of HIF-1α, phosphorylated phosphatidylinositol 3-hydroxy kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) proteins was determined by Western blotting (WB).
 Results: Compared with that in the control group, the number of tumors in the CC group was significantly increased (p < 0.05). Protein and messenger ribonucleic acid (mRNA) expressions of SOCS3 were down-regulated in CC group (p < 0.05), while protein expressions of p-PI3K, p-AKT and HIF-1α were significantly elevated in CC group (p < 0.05).
 Conclusion: SOCS3 is poorly expressed in CC rats, and promotes the expression of HIF-1α by activating PI3K/AKT signaling pathway. The findings, thus, provide a probable strategy for management of colon cancer.

Effect of electroacupuncture on protein expressions of SOCS3 and IRS-1 in hypothalamus and pancreas islet morphology in diabetic fatty rats

To observe the effect of electroacupuncture (EA) on protein expressions of suppressor of cytokine signaling 3 (SOCS3) and insulin receptor substrate-1 (IRS-1) in hypothalamus and morphology of pancreas islet in Zucker diabetic fatty (ZDF) rats, and to explore its possible mechanism on improving plasma glucose and insulin resistance of type 2 diabetes mellitus (T2DM). Twelve SPF male ZDF rats were selected and fed with high-fat diet for 4 weeks to establish the T2DM model, after modeling, the rats were randomly divided into a model group and an EA group, 6 rats in each one. Besides, 6 SPF male Zucker lean rats were selected as a blank group. In the EA group, EA was applied at "Pishu" (BL 20), "Weiwanxiashu" (EX-B 3), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), with continuous wave, 15 Hz in frequency, 2 mA in intensity, once a day, 20 min each time, 6 times a week for 4 weeks. The fasting plasma glucose (FPG) was measured before and after intervention. The serum level of fasting insulin (FINS) was measured by radioimmunoassay, and the homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated; the morphological change of pancreas islets was observed by HE staining; the protein expressions of SOCS3 and IRS-1 in hypothalamus were detected by Western blot. Before intervention, compared with the blank group, FPG in the model group and the EA group was increased (P<0.01). After intervention, compared with the blank group, FPG, serum level of FINS and HOMA-IR were increased (P<0.01), the protein expression of SOCS3 was increased while IRS-1 was decreased in the hypothalamus in the model group (P<0.01). Compared with the model group, FPG, serum level of FINS and HOMA-IR were decreased (P<0.01), the protein expression of SOCS3 was decreased while IRS-1 was increased in the hypothalamus in the EA group (P<0.01). In the model group, the shape of pancreas islets was irregular, the area of pancreas islets and the number of islet β cell nuclei were decreased, the nuclei of islet β cell was compensatory enlargement. In the EA group, the shape and the area of pancreas islets and the number of islet β cell nuclei were improved, the compensatory increase of islet β cell nuclei was alleviated compared with the model group. Electroacupuncture can reduce the fasting plasma glucose, improve the morphology of pancreas islets, and alleviate the insulin resistance in ZDF rats. The mechanism may relate to the down-regulation of SOCS3 and up-regulation of IRS-1 in the hypothalamus, and improving the function of hypothalamus in regulating peripheral glucose metabolism.

Expression of SOCS1 Protein in Endotoxin-Tolerant Mouse Model and Its Regulation Mechanism by mir-150.

In order to investigate the expression of Suppressor of Cytokine Signaling 1 (SOCS1) and its regulatory mechanism by mir-150 in a lipopolysaccharide (LPS) tolerant mouse model of endotoxin, a total of 60 male BALB/C mice were randomly divided into 2 groups. The LPS is used to construct the endotoxin resistant mouse model and the mice are included in the model group (n = 30), 0.9% sodium chloride injection is used to construct the normal control group (n = 30). And tumor necrosis factor-α (TNF-α) is determined by Elisa to determine whether the model was successfully constructed. The correlation between SOCS1 protein and mir-150 is analyzed by the Pearson correlation coefficient. In the experiments, the results show that the expression of TNF-α in the macrophage fluid of the model group is significantly decreased (P < 0.05), indicating that the endotoxin tolerance mouse model is successfully constructed, so the secretion of TNF-α is reduced.

Effects of SOCS3 on the development of colon cancer via regulation of HIF-1α

Purpose: To investigate the influence of suppressor of cytokine signaling 3 (SOCS3) on rats with colon cancer (CC).&#x0D; Methods: Sprague-Dawley (SD) rats were randomly divided into CC group and control group, and then CC rat model was constructed. The expression of SOCS3 in CC tissues was determined by quantitative real time-polymerase chain reaction (qRT-PCR). Hematoxylin-eosin staining (H&amp;E) was used to examine colon tissue morphology. Immunohistochemistry (IHC) staining assay was performed to determine the expression of SOCS3 protein in colon tissues. The contents of HIF-1α, phosphorylated phosphatidylinositol 3-hydroxy kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) proteins were determined by Western blotting (WB).&#x0D; Results: Compared with that in the control group, the number of tumors in CC group was significantly increased (p &lt; 0.05). 2). On the other hand, protein and message ribonucleic acid (mRNA) expressions of SOCS3 were down-regulated in CC group (p &lt; 0.05). 3), while protein expressions of p-PI3K, p-AKT and HIF-1α were raised in CC group (p &lt; 0.05).&#x0D; Conclusion: SOCS3 is lowly expressed in CC rats, and promotes the expression of HIF-1α by activating PI3K/AKT signaling pathway. Thus, SOCS3 provides a therapeutic strategy for the management of colon cancer.&#x0D; Keywords: Colon cancer; Suppressor of cytokine signaling protein 3 (SOCS3); Hypoxia inducible factor-1α

Long Non-coding RNA GAS5/miR-520-3p/SOCS3 Axis Regulates Inflammatory Response in Lipopolysaccharide-Induced Macrophages.

Sepsis is mainly caused by infection, and inflammation plays a vital role in the progression of sepsis. Increasing evidence shows the regulatory mechanism of long non-coding RNA growth arrest-specific 5 (GAS5) in inflammatory response. However, the potential role of GAS5 in sepsis was not really clear. Here, we set to investigate the role and mechanism of GAS5 in the inflammatory response of lipopolysaccharide (LPS)-induced macrophages in vitro. Levels of GAS5, microRNA-520-3p, suppressor of cytokine signaling 3 (SOCS3) and inflammatory cytokines tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 and IL-1β were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Nitric oxide (NO) release was measured through flow cytometry. The levels of TNF-α, IL-6, and IL-1β were also detected using ELISA. Dual-luciferase reporter and RNA pull-down assays were performed to clarify the relationship between miR-520-3p and GAS5 or SOCS3. Western blot was carried out to assess SOCS3 protein expression in macrophages. GAS5 level was remarkably decreased in sepsis serum and it was inversely related to the severity of sepsis. Upregulation of GAS5 repressed inflammatory response in LPS-induced macrophages, and the inhibitory effect was returned by miR-520-3p mimics. Moreover, miR-520-3p inhibitor downregulated the levels of inflammatory factors of TNF-α, IL-6, and IL-1β, as well as suppressed NO release. Mechanically, GAS5 functioned as a sponge of miR-520-3p and miR-520-3p directly targeted SOCS3. GAS5 regulated inflammatory response by the miR-520-3p/SOCS3 axis in LPS-induced macrophages, which furnished a novel therapeutic idea in clinical treatment of inflammation-induced sepsis.

SPTBN1 inhibits inflammatory responses and hepatocarcinogenesis via the stabilization of SOCS1 and downregulation of p65 in hepatocellular carcinoma.

Background: Spectrin, beta, non-erythrocytic 1 (SPTBN1), an adapter protein for transforming growth factor beta (TGF-β) signaling, is recognized as a tumor suppressor in the development of hepatocellular carcinoma (HCC); however, the underlying molecular mechanisms of this tumor suppression remain obscure.Methods: The effects on expression of pro-inflammatory cytokines upon the inhibition or impairment of SPTBN1 in HCC cell lines and liver tissues of Sptbn1+/- and wild-type (WT) mice were assessed by analyses of quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), enzyme linked immunosorbent assay (ELISA), Western blotting and gene array databases from HCC patients. We investigated the detailed molecular mechanisms underlying the inflammatory responses by immunoprecipitation-Western blotting, luciferase reporter assay, chromatin immunoprecipitation quantitative real time PCR (ChIP-qPCR), immunohistochemistry (IHC) and electrophoretic mobility shift assay (EMSA). The proportion of myeloid-derived suppressor cells in liver, spleen, bone marrow and peripheral blood samples from WT and Sptbn1+/- mice were measured by fluorescence-activated cell sorting (FACS) analysis. Further, the hepatocacinogenesis and its correlation with inflammatory microenvironment by loss of SPTBN1/SOCS1 and induction of p65 were analyzed by treating WT and Sptbn1+/- mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC).Results: Loss of SPTBN1 in HCC cells upregulated the expression of pro-inflammatory cytokines including interleukin-1α (IL-1α), IL-1β, and IL-6, and enhanced NF-κB transcriptional activation. Mechanistic analyses revealed that knockdown of SPTBN1 by siRNA downregulated the expression of suppressor of cytokine signaling 1 (SOCS1), an E3 ligase of p65, and subsequently upregulated p65 accumulation in the nucleus of HCC cells. Restoration of SOCS1 abrogated this SPTBN1 loss-associated elevation of p65 in HCC cells. In human HCC tissues, SPTBN1 gene expression was inversely correlated with gene expression of IL-1α, IL-1β and IL-6. Furthermore, a decrease in the levels of SPTBN1 gene, as well as an increase in the gene expression of IL-1β or IL-6 predicted shorter relapse free survival in HCC patients, and that HCC patients with low expression of SPTBN1 or SOCS1 protein is associated with poor survival. Heterozygous loss of SPTBN1 (Sptbn1+/-) in mice markedly upregulated hepatic expression of IL-1α, IL-1β and IL-6, and elevated the proportion of myeloid-derived suppressor cells (MDSCs) and CD4+CD25+Foxp3+ regulatory T cells (Foxp3+Treg) cells in the liver, promoting hepatocarcinogenesis of mouse fed by DDC.Conclusions: Our findings provided evidence that loss of SPTBN1 in HCC cells increases p65 protein stability via the inhibition of SOCS1 and enhances NF-κB activation, stimulating the release of inflammatory cytokines, which are critical molecular mechanisms for the loss of SPTBN1-induced liver cancer formation. Reduced SPTBN1 and SOCS1 predict poor outcome in HCC patients.

Long Non-Coding RNA NORAD Inhibits Breast Cancer Cell Proliferation and Metastasis by Regulating miR-155-5p/SOCS1 Axis

PurposeNon-coding RNA activated by DNA damage (NORAD) has been reported to be a cancer-related long non-coding RNA (lncRNA) implicated in the progression of several cancers; however, its role in breast cancer (BC) has not yet been clarified.MethodsQuantitative real-time polymerase chain reaction was used to examine NORAD, microRNA (miR)-155-5p, and suppressor of cytokine signaling 1 (SOCS1) mRNA expression levels. Western blotting was used to analyze SOCS1 protein expression. The malignancy of BC cells was assessed using the cell counting kit-8 (CCK-8), BrdU, and Transwell assays. Bioinformatics analysis, RNA immunoprecipitation assay, and dual-luciferase reporter gene assays were used to verify the targeted relationship between NORAD and miR-155-5p. Additionally, the regulatory effects of NORAD and miR-155-5p on SOCS1 expression were determined by western blotting.ResultsNORAD expression was significantly reduced in BC cell lines and tissues, and its low expression was associated with poor tumor tissue differentiation. NORAD overexpression repressed BC cell proliferation, migration, and invasion, whereas its knockdown produced the opposite effects. Additionally, miR-155-5p was found to be a target of NORAD, and the biological functions of miR-155-5p and NORAD were counteractive. MiR-155-5p was confirmed to target SOCS1, and SOCS1 was found to be positively regulated by NORAD.ConclusionNORAD suppresses miR-155-5p to upregulate SOCS1, thereby repressing the proliferation, migration, and invasion of BC cells.

Abstract 1709: New targeted therapy on SOCS1-related immune-response in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) includes heterogenous molecular subtypes, e.g., basal-like, immunomodulatory, mesenchymal, mesenchymal stem cell and luminal androgen receptor subtypes. Chemotherapy is the standard care for patients with early TNBC, but advanced or metastatic disease has unfavorable prognosis. PARP inhibitors and immune checkpoint inhibitors are effective for patients with BRCA gene mutation and PD-L1-positive TNBC, respectively. However, their clinical benefits seem to be limited. Thus, we are investigating new therapy targeted on immune modulation in TNBC. Materials and methods: First, we performed microarray expression analysis (Agilent Technologies, Inc.) of small RNA extracted from 11 breast cancer specimens. Second, miRNA subset was compared in TNBC and non-TNBC, and several genes were identified as predicted targets for interaction of miRNA in TNBC. Third, we examined a targeted gene and protein expressions in cancer tissues by real-time qPCR and immunohistochemical staining. Tissues samples were obtained from breast cancer patients with TNBC and non-TNBC, who were scheduled to receive neoadjuvant chemotherapy (NAC). Statistical significance is evaluated by chi-squared test. This study is approved at IRB in Kyorin University School of Medicine and also supported by Grant-in-Aid for Scientific Research C of Japan Society for the Promotion of Science. Results: From comprehensive analysis, SOCS1 (suppressor of cytokine signaling 1), IL-13 and several genes were predicted as interactive targets of miRNA in TNBC. We focused on SOCS1 gene amplification and its protein overexpression in breast cancer. Most patients were treated with NAC of anthracycline- and/or taxane-based regimens. Their pathological response was graded in surgical specimens according to classification of the Japanese Breast Cancer Society. From 55 tissue specimens stained by anti-SOCS1 antibody, there was no difference among staining intensity graded at score 0-3 and pathological response of chemotherapy. However, gene amplification of SOCS1 was not statistically different but relatively lower in 6 cases with pathologically partial or complete response (grade 2 or 3) than in 11 cases with no pathological response (grade 1a or 1b). Discussions: SOCS1 is well known as a negative regulator of JAK-STAT pathway. SOCS1 is also related to suppression of T cell activation and modulation of M1 macrophage. Our data suggest that down regulation of SOCS1 gene influences clinical benefit of chemotherapy as well as host immune response like tumor-infiltrating lymphocytes (TIL). We will examine relationship between SOCS1 protein expression and TIL in breast cancer and also examine cell proliferation and T cell activation in TNBC and non-TNBC cell lines under presence and absence of SOCS1 gene expression. Conclusion: SOCS1 is a promising molecular target to modulate immune editing in TNBC. Citation Format: Shigeru Imoto, Tomohiro Chiba, Sota Asaga, Hirotsugu Isaka, Shigehiro Yokoi, Yoshiharu Ishizaka, Ai Tsuchiya, Hiroshi Kamma. New targeted therapy on SOCS1-related immune-response in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1709.

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.

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.

Expression and methylation levels of suppressor of cytokine signaling 3 in rheumatic arthritis synovial fibroblasts

BackgroundIn the present study, we aimed to understand the expression and methylation levels of the suppressor of cytokine signaling 3 (SOCS3) in rheumatoid arthritis (RA) synovial fibroblasts. MethodThe RA model was established using Freund's complete adjuvant, and then the synovial fibroblasts were isolated and cultured. Next, RNA extraction and reverse transcription were performed. The SOCS3 transcription level was detected using qPCR, and SOCS3 protein expression was detected using western blotting (WB). Lastly, methylation-specific PCR (MSP) was performed. ResultsThe RA model was successfully demonstrated. SOCS3 gene (p < .01) and protein expression levels were significantly increased in the RA rat group compared to in the wild type (WT) group. However, no significant difference was observed in the MSP products between the RA and WT groups. ConclusionThe increased expression of the SOCS3 can be correlated with the development of RA.

Inhibition of miR‑155‑5p attenuates the valvular damage induced by rheumatic heart disease.

Autoimmunity is involved in the valvular damage caused by rheumatic heart disease (RHD). Increased evidence has linked microRNAs (miRNAs/miRs) to autoimmune disease. Signal transducer and activator of transcription 3 (STAT3) and sphingosine-1-phosphate receptor 1 (S1PR1) and suppressor of cytokine signaling 1 (SOCS1) have been widely studied for their roles in autoimmunity and inflammation. Thus, the current study aims to investigate the role played by miR-155-5p in RHD-induced valvular damage via the S1PR1, SOCS1/STAT3 and interleukin (IL)-6/STAT3 signaling pathways. An RHD rat model was induced by inactivated Group A streptococci and complete Freund's adjuvant. A recombinant adeno-associated virus (AAV-miR155-inhibitor) was used to inhibit the expression of miR-155-5p in the heart. Inflammation and fibrosis were assessed by hematoxylin and eosin staining and Sirius red staining. The expression of miR-155-5p in valvular tissues and serum exosomes was detected by reverse transcription-quantitative PCR. S1PR1, SOCS1, STAT3, phosphorylated STAT3, IL-6 and IL-17 protein expression was detected by western blotting and immunohistochemistry. The relationships between miR-155-5p and S1PR1 and SOCS1 were detected by dual luciferase assays. Cytokine concentrations were measured by ELISA. The expression of miR-155-5p in valve tissues and serum exosomes was increased along with decreased S1PR1 and activated SOCS1/STAT3 signaling in the RHD model. The expression of IL-6 and IL-17 was increased in the valves and the serum. Dual luciferase assays showed that miR-155-5p directly targeted S1PR1 and SOCS1. Inhibition of valvular miR-155-5p through AAV pretreatment increased S1PR1 expression and inhibited activation of the SOCS1/STAT3 signal pathway as a result of attenuated valvular inflammation and fibrosis as well as a decrease in IL-6 and IL-17 in the valves and serum. These results suggest that inhibition of miR-155-5p can reduce RHD-induced valvular damage via the S1PR1, SOCS1/STAT3 and IL-6/STAT3 signaling pathways.

Demethylation of SOCS1 mediates its abnormally high expression in ovarian cancer.

The present study aimed to investigate the association between methylation and the high expression of the suppressor of cytokine signaling 1 (SOCS1) in ovarian cancer by detecting the methylation rate and the degree of expression. The present study investigated the expression of SOCS1 mRNA and SOCS1 protein in ovarian cancer and normal ovary tissues using reverse transcription-quantitative polymerase chain reaction (PCR) and immunohistochemistry, and the methylation status of the CpG islands of SOCS1 mRNA in ovarian cancer tissue were examined using a methylation-specific PCR. The expression levels of SOCS1 mRNA in ovarian cancer specimens were significantly increased compared with that in the normal ovary tissues (P=0.0215). Consistent with this, the expression levels of SOCS1 protein in ovarian cancer specimens were significantly increased, while the methylation rate of SOCS1 mRNA was significantly decreased compared with that in the normal ovary tissues. Therefore, it may be concluded that the low methylation rate of SOCS1 mRNA in ovarian cancer increased the expression of SOCS1 mRNA, which may serve a role in the development of ovarian cancer.

LncRNA GAS5 promotes M1 macrophage polarization via miR-455-5p/SOCS3 pathway in childhood pneumonia.

We herein aimed to explore whether growth arrest-specific 5 (GAS5) promotes M1 macrophage polarization in childhood pneumonia and to investigate the underlying mechanism. Relative GAS5 and miR-455-5p expression and suppressor of cytokine signaling 3 (SOCS3) messenger RNA level were examined using quantitative reverse transcription polymerase chain reaction. Protein expression of SOCS3 and the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway-related proteins was detected using western blot analysis. Luciferase activity assay was performed to test whether miR-455-5p could bind to GAS5 or SOCS3. The macrophage phenotype was determined using flow cytometry analysis and enzyme-linked immunosorbent assay. The macrophage polarization toward the M2 phenotype was observed in peripheral blood from pneumonia children. Furthermore, GAS5 and SOCS3 expression were upregulated but miR-455-5p downregulated in human monocyte-derived macrophages from pneumonia children compared with the control group. Furthermore, GAS5 acted as a sponge for miR-455-5p to facilitate SOCS3 expression. Moreover, miR-455-5p mimic and SOCS3 knockdown significantly reversed the GAS5 overexpression-mediated suppression of the JAK2/STAT3 signaling and promotion of M1 polarization. GAS5 promotes M1 macrophage polarization by acting as a competing endogenous RNA of miR-455-5p to facilitate SOCS3 expression in childhood pneumonia.

Expression of SOCS1 in overload papillary muscles of mice cardiac ventricle

Objective To investigate the expression of suppressor of cytokine signaling 1 (SOCS1) in overloaded ventricle papillary muscle, so as to understand its expression characteristics in structural remodeling after the overloading and the biomechanical properties of the muscle under cubic jellyfish toxin-1(CfTX-1) pretreatment that can affect cell signal transduction. Methods Abdominal aortic-venous fistula (AVF) were operated in Kunming mice (n=5), and the cardiac left ventricles were harvested after two weeks of fistulation. The mice in normal group were sham operated as a control (n=5). In vitro culture, the left ventricular papillary muscle of normal mice was used (n=20). In the stretching group, the isolated papillary muscles were double-ratio stretched and fixed on silicone plate. In the relaxation group, the muscles were not stretched. A separated subgroup that transfected with SOCS1 plasmids were set in each group of stretching and relaxation. The papillary muscle samples of each group were cultured in culture medium for 3 days at 37 ℃, and then homogenized for extracting total protein. The total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 23 ku band with SOCS1 was used as the target band, and the integrated optical density (IOD) value was measured by computer image analysis method. The expression of SOCS1 protein was detected by Western Blot and the imprinted IOD value was also measured. The papillary muscle in the stretching group was stretched by micro-positioned stretching method, and the initial load was 1 g. After stabilization, the papillary muscle was stretched by 15 mm for continuously 5 times, and the passive tension characteristic curves during the first and fifth stretching were observed and recorded. The peak passive tension (PTmax) and its deceleration velocity (DV) of the papillary muscle were calculated based on the curves. Results Comparing with the AVF group, the normal group had higher IOD values of 23 ku band and SOCS1 blot in total protein of the papillary muscle, and the differences were statistically significant (all P<0.01). The IOD value of 23 ku band in the SOCS1 transfected stretching group was significantly higher than those of the two relaxation groups, and the differences were statistically significant (all P<0.01). However, the difference of this value was not statistically significant between the two relaxation groups. The average IOD value of SOCS1 blot in the SOCS1 transfected stretching group was higher than those of the normal stretching group and the SOCS1 transfected relaxation group, and the differences were statistically significant (all P<0.01). Comparing with the normal group, the AVF group had higher PTmax and ultimate PTmax of the papillary muscles, and had a lower DV values, and the differences were statistically significant (all P<0.01). Conclusions The expression of SOCS1 is sensitive to tension load, and has a positive effect as an overload-sensitive signal in improving myocardial adaptability, protecting myocardial structure and maintaining systolic and diastolic function. CfTX-1 also has a positive effect on improving the compliance of ventricular papillary muscles. Key words: Mechanical load; Ventricular papillary muscle; Stretching culture; Transfection; Suppressor of cytokine signaling 1

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.

PPARγ alleviated hepatocyte steatosis through reducing SOCS3 by inhibiting JAK2/STAT3 pathway

Peroxisome proliferator-activated receptor gamma (PPARγ) participates in the process of insulin resistance (IR), a crucial pathophysiology in non-alcoholic fatty liver disease (NAFLD). Meanwhile, suppressor of cytokine signaling3 (SOCS3) also regulates IR in NAFLD. Both PPARγ and SOCS3 play a role in NAFLD through regulating IR, while it is unclear whether these two proteins interact to regulate hepatic steatosis. PPARγ, SOCS3 and its associated JAK2/STAT3 pathway were analyzed using Kuppfer cells (KCs) treatment with LPS and BRL-3A cells treatment with palmitic acid, KC-conditioned medium (KCCM), PPARγ agonist rosiglitazone (ROZ) or JAK2 inhibitor AG490 to demonstrate the role of PPARγ and SOCS3 in hepatocytes steatosis. As LPS concentration increasing, phagocytosis activity of KCs decreased; but releasing of TNF-α and IL-6 increased. After treatment with KCCM, mRNA level of SOCS3, JAK2 and STAT3 as well as protein expression of SOCS3, p-JAK2 and p-STAT3 in steatosis BRL-3A cells increased significantly, which were inhibited by AG490 or ROZ treatment. Taken together, these results indicated that KCCM attributed to KCs dysfunction facilitated hepatocyte steatosis through promoting expressing SOCS3; but PPARγ agonist ROZ alleviated steatosis through reducing SOCS3 expression by inhibiting JAK2/STAT3 in hepatocytes.