Suppressor Of Cytokine Signaling 3 mRNA 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.

N6-methyladenosine (m6A) writer METTL3 accelerates the apoptosis of vascular endothelial cells in high glucose

Recent studies have shown that N6-methyladenosine (m6A) methylation, one of the most prevalent epigenetic modifications, is involved in diabetes mellitus. However, whether m6A regulates diabetic vascular endothelium injury is still elusive. Present research aimed to investigate the regulation and mechanism of m6A on vascular endothelium injury. Upregulation of METTL3 was observed in the high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs), following with the upregulation of m6A methylation level. Functionally, METTL3 silencing repressed the apoptosis and recovered the proliferation of HUVECs disposed by HG. Moreover, HG exposure upregulated the expression of suppressor of cytokine signaling3 (SOCS3). Mechanistically, METTL3 targeted the m6A site on SOCS3 mRNA, which positively regulated the mRNA stability of SOCS3. In conclusion, METTL3 silencing attenuated the HG-induced vascular endothelium cells injury via promoting SOCS3 stability. In conclusion, this research expands the understanding of m6A on vasculopathy in diabetes mellitus and provides a potential strategy for the protection of vascular endothelial injury.

MiR-210-3p Promotes Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Targeting SOCS1-Mediated NF-κB Pathway.

Under the condition of chronic obesity, an increased level of free fatty acids along with low oxygen tension in the adipose tissue creates a pathophysiological adipose tissue microenvironment (ATenv), leading to the impairment of adipocyte function and insulin resistance. Here, we found the synergistic effect of hypoxia and lipid (H + L) surge in fostering adipose tissue macrophage (ATM) inflammation and polarization. ATenv significantly increased miR-210-3p expression in ATMs which promotes NF-κB activation-dependent proinflammatory cytokine expression along with the downregulation of anti-inflammatory cytokine expression. Interestingly, delivery of miR-210-3p mimic significantly increased macrophage inflammation in the absence of H + L co-stimulation, while miR-210-3p inhibitor notably compromised H + L-induced macrophage inflammation through increased production of suppressor of cytokine signaling 1 (SOCS1), a negative regulator of the NF-κB inflammatory signaling pathway. Mechanistically, miR-210 directly binds to the 3'-UTR of SOCS1 mRNA and silences its expression, thus preventing proteasomal degradation of NF-κB p65. Direct delivery of anti-miR-210-3p LNA in the ATenv markedly rescued mice from obesity-induced adipose tissue inflammation and insulin resistance. Thus, miR-210-3p inhibition in ATMs could serve as a novel therapeutic strategy for managing obesity-induced type 2 diabetes.

Downregulation of miR-122 by porcine reproductive and respiratory syndrome virus promotes viral replication by targeting SOCS3

MicroRNAs are small non-coding RNA that regulate host anti-viral immune response. In this study, we used high-throughput sequencing to identify miRNAs that were differentially expressed upon PRRSV infection in porcine alveolar macrophages. We observed that the expression level of miR-122 was decreased upon PRRSV infection. Over-expression of miR-122 remarkably suppressed PRRSV replication, while blockage of endogenous miR-122 enhanced PRRSV replication. Moreover, over-expression of miR-122 reduced the protein level of porcine suppressor of cytokine signaling 3 (SOCS3), a negative regulator of JAK-STAT signaling, resulting in enhanced production of type Ⅰ IFN. Further analysis revealed that miR-122 decreased the expression of SOCS3 at the post-transcription level by targeting the 3’ UTR region of SOCS3 mRNA. In conclusion, this study demonstrates that the expression of miR-122 was reduced during PRRSV infection. miR-122 impaired PRRSV replication by promoting the production of type I interferon. Our study may provide new insights into understanding PRRSV immune evasion mechanisms.

CpG Island Methylation of Suppressor of Cytokine Signaling-1 Gene Induced by HCV Is Associated With HCV-Related Hepatocellular Carcinoma.

Suppressor of cytokine signaling 1 (SOCS-1) is implicated in both virus infection and carcinogenesis. This study investigated the role of HCV infection on SOCS-1 in normal and HCV-infected tissues and revealed a possible mechanism underlying HCV-induced hepatocellular carcinoma (HCC) genesis. In total, 10 HCV-HCC tissues, seven adjacent tissues, seven distal tissues, and 16 normal liver tissues were collected. SOCS-1 expression in tissue sections was detected by immunohistochemistry. After viral load was quantified, the correlation between SOCS-1 expression and viral load was analyzed in different tissues. Then, HCV replicon model was used to detect a relationship between HCV and SOCS-1. Subsequently, methylation-specific PCR (MSP) was applied to show the methylation status of SOCS-1 genes in normal tissues and HCV-replicating cell lines. A correlation between gene methylation, SOCS-1 expression, and HCV was analyzed. The lowest expression of SOCS-1 was observed in HCV-HCC tissues. Tissues with a higher HCV viral load showed lower SOCS-1 expression (p = 0.0282). Consistently, SOCS-1 mRNA and protein were lower in HCV-replicating cell lines than in uninfected ones. Furthermore, gene methylation was found in all examined tissues but higher in HCC tissues, and it is positively correlated with HCV viral load (r2 = 0.7309, p < 0.0001). HCV infection would upregulate methylation of the SOCS-1 gene in HCV-replicating cell lines. The downregulation of SOCS-1 in normal and HCV-replicating cell lines may result from HCV infection through epigenetic regulation, in which gene methylation in the CpG island of SOCS-1 promoters upon HCV infection suppresses its expression.

MiR-221 Regulates Suppressors of Cytokine Signaling 3-Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (SOCS3-JAK2/STAT3) Pathway and Affects Thyroid Cancer Cell Proliferation and Apoptosis

Objective: Suppressors of cytokine signaling 3 (SOCS3) negatively regulates JAK-STAT signaling. Bioinformatics analysis showed a targeted relationship between miR-221 and SOCS3 mRNA 3′-UTR. This study investigated whether miR-221 regulates SOCS3 expression and affects thyroid cancer cells. Methods: Dual-luciferase reporter gene experiments verified the relationship between miR-221 and SOCS3. The tumor tissues and adjacent tissues of patients with thyroid cancer were collected to detect miR-221 and SOCS3 level. Thyroid cancer cell line KTC-1 cells were assigned into miR-NC group and miR-221 inhibitor group followed by analysis of SOCS3, p-JAK2, and p-STAT3 level by Real-time PCR, cell apoptosis and cell proliferation by flow cytometry and cell invasion by Transwell assay. Results: Compared with adjacent tissues, miR-221 level in tumor tissues was increased, and SCOS3 mRNA level was decreased. There was a targeted relationship between miR-221 and SOCS3 mRNA. MiR-221 level in KTC-1 and TPC-1 cells was increased, while SOCS3 mRNA level was decreased. MiR-221 inhibitor can significantly upregulate SOCS3 mRNA and protein in KTC-1 cells, reduce the expression of p-JAK2, p-STAT3 protein, increase cell apoptosis, and reduce cell proliferation and invasion. Conclusion: The increased miR-221 and decreased SOCS3 expression are related to thyroid cancer pathogenesis. MiR-221 can inhibit the expression of SOCS3, affect JAK-STAT signaling activity, and regulate the proliferation and apoptosis of thyroid cancer cells.

YTHDF1 Negatively Regulates Treponema pallidum-Induced Inflammation in THP-1 Macrophages by Promoting SOCS3 Translation in an m6A-Dependent Manner.

BackgroundPrevious studies have confirmed that the bacterium Treponema pallidum (TP) or its proteins provide signals to macrophages that induce an inflammatory response; however, little is known about the negative regulation of this macrophage-mediated inflammatory response during syphilis infection or the underlying mechanism. Recent evidence suggests the role of the RNA modification, N6-adenosine methylation (m6A), in regulating the inflammatory response and pathogen-host cell interactions. Therefore, we hypothesized that m6A plays a role in the regulation of the inflammatory response in macrophages exposed to TP.MethodsWe first assessed m6A levels in TP-infected macrophages differentiated from the human monocyte cell line THP-1. The binding and interaction between the m6A “writer” methyltransferase-like 3 (METTL3) or the m6A “reader” YT521-B homology (YTH) domain-containing protein YTHDF1 and the suppressor of cytokine signaling 3 (SOCS3), as a major regulator of the inflammatory response, were explored in differentiated TP-infected THP-1 cells as well as in secondary syphilitic lesions from patients. The mechanisms by which YTHDF1 and SOCS3 regulate the inflammatory response in macrophages were assessed.Results and ConclusionAfter macrophages were stimulated by TP, YTHDF1 was upregulated in the cells. YTHDF1 was also upregulated in the syphilitic lesions compared to adjacent tissue in patients. YTHDF1 recognizes and binds to the m6A methylation site of SOCS3 mRNA, consequently promoting its translation, thereby inhibiting the JAK2/STAT3 pathway, and reducing the secretion of inflammatory factors, which results in anti-inflammatory regulation. This study provides the first demonstration of the role of m6A methylation in the pathological process of syphilis and further offers new insight into the pathogenesis of TP infection.

Expression Level of SOCS3 in Acute Lymphoblastic Leukemia Cells Affects the Cytotoxicity of NK Cells

To detect the expression level of suppressors of cytokine signaling 3 （SOCS3） in acute lymphoblastic leukemia (ALL), and to observe the effect of over-expresson of SOCS3 in Jurkat cells on the cytotoxicity of NK cells. The expression levels of SOCS3 mRNA in peripheral blood mononuclear cells of 20 children with ALL and 20 healthy children (normal control group) were detected by RT-PCR. The peripheral blood NK cells from healthy subjects were selected by immunomagnetic technique, and the purity was detected by flow cytometry. SOCS3 was overexpressed in Jurkat cells infected with lentivirus vector, and SOCS3 mRNA expression was detected by RT-PCR after lentivirus infection. The NK cells were co-cultured with the infected Jurkat, and LDH release method was used to detect the cytotoxicity of NK cells on the infected Jurkat cells. The concentrations of TNF-α and IFN-γ were determined by ELISA. The expression of NKG2D ligands MICA and MICB on the surface of Jurkat cells were detected by flow cytometry. Western blot was used to detect the effect of SOCS3 overexpression on STAT3 phosphorylation in Jurkat cells. Compared with the control group, the mRNA expression of SOCS3 in the peripheral blood mononucleated cells of ALL children was significantly decreased. The purity of NK cells isolated by flow cytometry could reach more than 70%. The expression of SOCS3 mRNA in Jurkat cells increased significantly after lentivirus infection. Overexpression of SOCS3 in Jurkat cells significantly promoted the killing ability of NK cells and up-regulated the secretion of TNF-α and IFN-γ from NK cells. The results of flow cytometry showed that the expression of NKG2D ligands MICA and MICB on Jurkat cells increased significantly after SOCS3 overexpression. Western blot results showed that overexpression of SOCS3 significantly reduced the phosphorylation level of STAT3 protein in Jurkat cells. SOCS3 mRNA expression was significantly decreased in ALL patients, and overexpression of SOCS3 may up-regulate the expression of MICA and MICB of NKG2D ligands on Jurkat cell surface through negative regulation of JAK/STAT signaling pathway, thereby promoting the cytotoxic function of NK cells.

SOCS3 Gene Polymorphism and Hypertension Susceptibility in Chinese Population: A Two-Center Case-Control Study.

Endothelial inflammation and vascular damage are essential risk factors contributing to hypertension. Suppressor of cytokine signaling 3 (SOCS3) is involved in the regulation of multiple inflammatory pathways. A large number of studies have shown that the anti-inflammatory effect of SOCS3 in hypertension, obesity, and allergic reactions has brought more insights into the inhibition of inflammation. Therefore, we selected a tagSNP of SOCS3 (rs8064821) to investigate whether they are contributing to the risk of hypertension in the Chinese population. In total, 532 patients with hypertension and 569 healthy controls were enrolled for two central of China. SOCS3 rs8064821 C>A polymorphism was genotyped using TaqMan assay. SOCS3 rs8064821 CA genotype was associated with an increased risk of hypertension (OR = 1.821, 95%CI = 1.276-2.600, P = 0.001). Rs8064821 A allele was associated with higher SOCS3 mRNA level in PBMCs from healthy donors. SOCS3 rs8064821 C>A polymorphism may contribute to the risk of hypertension in the Chinese population by regulating the expression of SOCS3.

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.

Interleukin-24 as a Pulmonary Target Cytokine in Bronchopulmonary Dysplasia.

The proliferation of fetal alveolar type II cells (FATIICs) was impaired in bronchopulmonary dysplasia (BPD), which is modulated by hyperoxia and inflammatory response. Interleukin 24 (IL-24), a cytokine produced by certain cell types, plays an essential role in inflammation and host protection against infection. However, the ability of FATIICs to produce IL-24 remains unclear, and the role of IL-24 in BPD progression is yet to be determined. With reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay, the authors evaluated whether FATIICs produce IL-24 in physiological conditions. The authors quantified IL-24 expression in the lungs of newborn rat pups exposed to hyperoxia (70% oxygen) and in FATIICs isolated on embryonic day 19 that were exposed to 95% oxygen or lipopolysaccharide (LPS). The role of IL-24 in FATIICs, cell proliferation, cell apoptosis, and cell cycle were further evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometric analysis. Also, they assessed caspase-3 and SOCS3 mRNA in IL-24 siRNA-treated cells by using RT-qPCR. During culture, IL-24 mRNA and protein levels in FATIICs gradually decreased with FATIIC differentiation. IL-24 expression increased significantly in rat lungs exposed to hyperoxia and FATIICs exposed to oxygen or LPS. Recombinant IL-24 enhanced cell proliferation by decreasing the proportion of apoptotic cells and increasing the proportion of cells in the S phase. The IL-24 siRNA-treated cells expressed more caspase-3 mRNA. Furthermore, suppressor of cytokine signaling 3 (SOCS3) mRNA was significantly decreased in rats and FATIICs exposed to oxygen, whereas it dramatically increased in FATIICs exposed to LPS. The IL-24 siRNA-treated cells expressed more SOCS3 mRNA. These studies suggest IL-24 is a pulmonary target cytokine in BPD, and may possibly regulate SOCS3 in oxidative stress and inflammation of the lung.

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.

M6A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

N6-methyladenosine (m6A) RNA modification maintained by N6-methyltransferases and demethylases is involved in multiple biological functions. Methyltransferase like 3 (METTL3) is a major N6-methyltransferase. However, the role of METTL3 and its installed m6A modification in colorectal tumorigenesis remains to be fully elucidated. METTL3 is highly expressed as indicated in colorectal cancer samples in the TCGA and Oncomine databases, implying its potential role in colon tumorigenesis. SW480 cell line with stable METTL3 knockout (METTL3-KO) was generated using CRISPR/Cas9 and were confirmed by the loss of METTL3 expression and suppression of m6A modification. The proliferation of METTL3-KO cells was significantly inhibited compared with that of control cells. METTL3-KO decreased the decay rate of suppressor of cytokine signaling 2 (SOCS2) RNA, resulting in elevated SOCS2 protein expression. m6A-RNA immunoprecipitation-qPCR (MeRIP-qPCR) revealed that SOCS2 mRNA was targeted by METTL3 for m6A modification. Similar to METTL3-KO SW480 cells, SW480 cells treated with 3-deazaadenosine, an RNA methylation inhibitor, exhibited elevated SOCS2 protein expression. Increased levels of SOCS2 in METTL3-KO SW480 cells were associated with decreased expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), contributing to the inhibition of cell proliferation. The underlying associations among METTL3, SOCS2, and LGR5 were further confirmed in SW480 cells transfected with si-METTL3 and in tumor samples from patients with colorectal cancer. Taken together, our data demonstrate that an increased level of METTL3 may maintain the tumorigenicity of colon cancer cells by suppressing SOCS2.

MicroRNA-221 (Mir-221) Influences Ovarian Cancer Cell Proliferation and Apoptosis Through Regulating Suppressors of Cytokine Signaling 3-Janus Kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) Pathway

Suppressors of cytokine signaling 3 (SOCS3) regulates JAK-STAT signaling. Bioinformatics analysis showed a targeted relationship of Mir-221 with SOCS3 mRNA. Our study assessed whether Mir221 regulates SOCS3 expression and affects ovarian cancer cells. Ovarian cancer tissues were collected and compared with adjacent tissues to detect Mir-221 and SOCS3 expression. Ovarian cancer cell line SKOV3 cells were separated into miR-NC group and Mir-221 inhibitor group followed by analysis of Mir-221, SOCS3, p-JAK2, and p-STAT3 expression, cell apoptosis and proliferation. Compared with adjacent tissues, Mir-221 expression in tumor tissues was significantly elevated and SCOS3 mRNA level was decreased. There is a targeted relationship between miR-203 and SOCS3 mRNA. Compared with IOSE80 cells, ovarian cancer A2780 and SKOV3 cells presented significantly elevated Mir-221 level and decreased SOCS3 expression. Mir-221 inhibitor transfection significantly upregulated SOCS3, downregulated p-JAK2, p-STAT3 protein, promoted cell apoptosis and inhibited proliferation. The increased Mir-221 and decreased SOCS3 expression are related to the pathogenesis of ovarian cancer. Mir-221 downregulates SOCS3, affects the activity of JAK-STAT signaling, and regulates ovarian cancer cell proliferation and apoptosis.

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.

Effects of miR-150-5p on the growth and SOCS1 expression of rheumatoid arthritis synovial fibroblasts.

miR-150-5p has been implicated in the regulation and onset of immune diseases. We investigated the effects of miR-150-5p on the functions of RA synovial fibroblasts (RASFs). The binding site between suppressor of cytokine signaling 1 (SOCS1) and miR-150-5p was analyzed using European Bioinformatics Institute database, and the 3' UTR of SOCS1 mRNA, including the binding site, was amplified and ligated to the 3'-end of LUC2 gene in the pmirGL0 dual-luciferase vector. The pmirGL0 vector and corresponding mimics were subsequently co-transfected into 293T cells to compare the relative fluorescence intensity of LUC2 between the miR-150-5p mimics and the negative control (NC) mimics groups. Further, the RASF cell line MH7A was transfected with miR-150-5p or NC mimics and subjected to flow cytometric analysis, cell counting kit-8 assay, western blot analysis, qPCR, and enzyme-linked immunosorbent (ELISA) assay 48h after transfection. miR-150-5p mimics resulted in a lower cell apoptotic rate and proportion of cells in the S phase. Using a dual-luciferase reporter gene assay, we then found that SOCS1 is a potential target of miR-150-5p. Compared with NC mimics, miR-150-5p mimics significantly decreased the protein and mRNA expression levels of SOCS1. ELISA assay showed that miR-150-5p mimics increased interleukin-6 level in the cell culture medium but did not influence tumor necrosis factor-alpha levels. Overall, the growth-promoting effect of miR-150-5p on MH7A cells may be attributed to the miR-150-5p-induced degradation of SOCS1 mRNA, suggesting a potential therapeutic target for RA.Key Points• SOCS1 is a potential target of miR-150-5p.• miR-150-5p promoted the growth of RASF cell line MH7A.• miR-150-5p increased the secretion of IL-6 but did not significantly affect TNF-α levels in MH7A cells.

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.