Signal Transducer And Activator Of Transcription 3 mRNA Research Articles

Mechnism of CD4+CD25+ forkhead box P3+ regulatory T cells suppressing the tumor immunity in colorectal cancer

Objective To study the mechanism of CD4 + CD25 + forkhead box P3 (Foxp3) + regulatory T cells inhibiting the tumor immunity in colorectal cancer.Methods Thirty-two cases of colorectal carcinoma,21 cases of polypus,and 18 cases of normal tissues were collected.T he immunohistochemistry was used to measure the number of Foxp3 + regulatory T cells and interleukin (IL)-10 positive cells in above tissues,and reverse transcription-polymerase chain reaction (RT-PCR) was done to dectect the expression of Foxp3 and signal transducers and activators of transcription 3 (STAT3) mRNA in different tissues.The relationship between the above indexes and clinicopathological features was analyzed.Results The expression rate of positive cells of Foxp3 and IL-10 in colorectal cancer was respectively 45.8％ and 68.8％,significantly higher than in the polypus tissues (13.6％ and 1.8％) and normal tissues (9.5％and 23.8％) (P ＜0.01 for all).The relative expression amount of Foxp3 and STAT3 mRNA in tumor tissue was respectively 0.644 ± 0.059 and 0.343 ± 0.036,which was significantly increased as compared with polypus (0.322 ±0.020 and 0.212 ±0.028) and normal tissue (0.309 ±0.016 and 0.202 ±0.021)(P ＜0.01 for all).The Foxp3 mRNA expression in colorectal cancer tissues was positively correlated with STAT3 (r =0.526.P ＜ 0.05).The Foxp3 protein expression wass positively correlated with IL-10 (r =0.314,P ＜ 0.05).The expression of Foxp3 in cancer patients was visibley correlated with the histological grade,lymph node metastasis and TNM stage (P ＜ 0.05),and there was no visible correlation with age and gender (P ＞ 0.05).Conclusion CD4 + CD25 + Foxp3 + regulatory T cells inhibit tumor immunity and induce tumor immune escape through the expression of Foxp3 and some other related inhibitory cytokines,and finally promote the tumor progression. Key words: Regulatory T cell; Forkhead box P3; Signal transducers and activators of transcription 3 ; Colorectal cancer; Tumor immunity

Synthesis and Hybridization Studies of a New CPP-PNA Conjugate as a Potential Therapeutic Agent in Atherosclerosis Treatment

The objective of this study was to design and synthesize a new CPP-PNA conjugate that would be able to penetrate endothelial cells, bind STAT1 mRNA and thereby block the activity of STAT1 (the Signal Transducer and Activator of Transcription 1), which is important in cases of vessel inflammation. In the course of the study, the TAMRA-PTD-4- Hal(traziole-Gly-PNA)-conjugate was successfully synthesized using a specific 1,3-dipolar Huisgen cycloaddition reaction known as a "click reaction". The hybridization properties of the conjugate to complementary hSTAT1 mRNA and hSTAT1 ssDNA fragments was verified by capillary electrophoresis (CE). Studies have shown that the attachment of a fluorescence-labeled peptide to a PNA sequence via a 1,2,3-triazole ring did not alter the binding properties of the PNA to the complementary hSTAT1 mRNA or hSTAT1 ssDNA fragments maintaining similar binding affinity. Furthermore, the data obtained suggest that the use of such a conjugate to modulate the activity and expression of STAT1 could provide a new therapeutic strategy for atherosclerosis treatment.

Role of Unphosphorylated Transcription Factor STAT3 in Late Cerebral Ischemia after Subarachnoid Hemorrhage

Molecular mechanisms behind increased cerebral vasospasm and local inflammation in late cerebral ischemia after subarachnoid hemorrhage (SAH) are poorly elucidated. Using system biology tools and experimental SAH models, we have identified signal transducer and activator of transcription 3 (STAT3) transcription factor as a possible major regulatory molecule. On the basis of the presence of transcription factor binding sequence in the promoters of differentially regulated genes (significant enrichment PE: 6 × 10(5)) and the consistent expression of STAT3 (mRNA, P=0.0159 and Protein, P=0.0467), we hypothesize that unphosphorylated STAT3 may directly DNA bind and probably affect the genes that are involved in inflammation and late cerebral ischemia to influence the pathologic progression of SAH.

The effects of STAT3 and Survivin silencing on the growth of human bladder carcinoma cells

Despite accumulating evidence suggesting a critical role of signal transducer and activator of transcription 3 (STAT3) and Survivin in human bladder cancer, the effects of silencing these genes on the proliferation of T24 bladder carcinoma cells remain unknown. Here, we investigated the inhibitory effects of STAT3 or Survivin silencing on the in vitro and in vivo growth of human T24 bladder carcinoma cells. Small interfering RNA (siRNA) vectors targeting STAT3, Survivin, or both genes were designed and synthesized. The recombinant plasmid DNA constructs were confirmed by DNA sequencing. They were then transiently transfected into T24 cells, and the mRNA and protein expressions of STAT3 and Survivin were determined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot, respectively. Cell proliferation was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The in vivo growth-inhibition effects of the siRNA vectors were assessed using a bladder cancer mouse model. The tumor weight and size was recorded 4 weeks post-inoculation. We successfully synthesized four siRNA vectors targeting STAT3 and four vectors targeting Survivin. The STAT3-3 and Survivin-4 siRNA constructs were most efficient in reducing STAT3 or Survivin expression, respectively. We then constructed a vector containing these two sequences together (STAT3-Survivin siRNA) and found that the single vector efficiently silenced STAT3 and Survivin expression. Moreover, silencing of STAT3 or Survivin significantly suppressed the in vitro and in vivo proliferation of T24 cells compared to the controls (P<0.05). Our findings indicated that the downregulation of STAT3 or Survivin can suppress the proliferation of T24 bladder cancer cells. Moreover, no additive effects were observed when STAT3 and Survivin were knocked down together, suggesting that they work in the same signaling pathway in T24 cells. These results provide valuable insights into understanding the pathways involved during the tumorigenesis of bladder cancer.

MiR-124 functions as a tumor suppressor in the endometrial carcinoma cell line HEC-1B partly by suppressing STAT3

MicroRNAs (miRNAs) play an important role in the development and progression of endometrial carcinoma (EC). Recently, several studies have shown that microRNA-124 (miR-124) is downregulated in various cancers, which can affect tumor initiation and maintenance. However, the effects of miR-124 on EC are largely unknown. In this study, we identified the under-expression of miR-124 in 35 paired EC tissues and adjacent normal tissues. Further, functional experiments found that ectopic expression of miR-124 markedly suppressed cell proliferation, migration, and invasion of EC cells. It also induced cell apoptosis and G1-phase cell cycle arrest. Moreover, we identified signal transducer and activator of transcription 3 (STAT3) as a direct target of miR-124, and over expression of miR-124 not only induced changes in STAT3 expression but also altered expression of its target genes, cyclin D2 and matrix metalloproteinase 2, in the human endometrial carcinoma cell line HEC-1B. In addition to targeting STAT3 directly, we found that miR-124 suppresses phosphorylation of STAT3 through targeting IL-6R indirectly. Restored STAT3 expression through treatment with IL-6 cytokine partly abolished miR-124-mediated cell cycle arrest and apoptosis induction. These results combined with the tumorigenetic role of STAT3 in HEC-1B cells suggest that the antitumor effects of miR-124 are achieved, at least partly, through down regulation of STAT3 mRNA and its downstream target genes. Therefore, inhibition of constitutively activated STAT3 by ectopic expression of miR-124 in EC may provide a novel therapeutic strategy for the treatment of EC.

Effects of sevoflurane and isoflurane on proliferation of neural stem cells in rat cerebral cortex

Objective To evaluate the effects of sevoflurane and isoflurane on the proliferation of neural stem cells in rat cerebral cortex.Methods The neural stem cells were isolated from Sprague-Dawley rats at 15 days of gestation and cultured at a density of (1-2) × 106 cells/ml.The cells of 3rd generation were seeded in 6-well plates coated with poly-lysine and randomly divided into 3 groups (n =24 wells each) using a random number table:control group (group C),sevoflurane group (group S) and isoflurane group (group Ⅰ).In S and Ⅰ groups,the cells were exposed to 4.9％ sevoflurane and 2.8％ isoflurane in a mixture of 5％ CO2-95％ O2 for 6 h,respectively.The cells were exposed to a mixture of 5 ％ CO2-95 ％ O2 for 6 h in group C.After 6 h of exposure,the plates were removed and the cells were continuously incubated for 2 h in an incubator at 37 ℃.The proliferation of cells was detected by immunocytochemistry and microplate method.The expression of proliferation-related genes such as signal transducers and activators of transcription 3 (STAT3),Sox2,Notchl and P21 mRNA was detected using quantitative real-time PCR.The expression of total STAT3 protein and phosphorylated STAT3 protein (p-STAT3) was determined using Western blot.Results Compared with C group,the rate of proliferation was significantly decreased,and the expression of p-STAT3 was down-regulated in I and S groups,and the expression of STAT3 mRNA was down-regulated in Ⅰ group (P ＜ 0.05),and no significant change was found in the expression of STAT3 mRNA in S group (P ＞ 0.05).There was no significant difference in the expression of Sox2,Notch1 and P21 mRNA and total STAT3 protein between the three groups (P ＞ 0.05).Conclusion Sevoflurane and isoflurane both can inhibit the proliferation of neural stem cells in the rat cerebral cortex,and the mechanism may be that sevoflurane inhibits activation of STAT3 protein,however,isoflurane not only inhibits the activation of STAT3 protein,but also inhibit the transcription of STAT3 gene. Key words: Isoflurane; Stem cells; Neural stem cells; Cell proliferation; Sevoflurane

Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells

MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL. Disclosures:No relevant conflicts of interest to declare.

Role of STAT3 signal transduction pathway in diazoxide cardioplegic solution-induced reduction of ischemia-reperfusion injury in isolated rat hearts

Objective To evaluate the role of signal transducer and activator of transcription 3 (STAT3) signal transduction pathway in diazoxide cardioplegic solution-induced reduction of ischemia-reperfusion (I/R) injury in isolated rat hearts.Methods Sixty adult male Sprague-Dawley rats,aged 2-3 months,weighing 240-260 g,were used in this study.Their hearts were excised and perfused in a Langendorff apparatus and then randomly divided into 5 groups (n=12 each):control group (group C),group I/R,cardioplegic solution group (group P),diazoxide cardioplegic solution group (group DZX),and STAT3 signal transduction pathway blocker Stattic group (group Stattic).The hearts were continuously perfused for 90 min after 15 min of equilibration in group C.Perfusion was stopped after 15 min of equilibration and restored 30 min later in I/R,P,DZX and Stattic groups.In P and DZX groups,the hearts were perfused with the cardioplegic solution containing 0.4％ dimethyl sulfoxide and 50μmol/L diazoxide,respectively,before perfusion was stopped.In group Stattic,the hearts were perfused with 10μmol/L Stattic for 5 min before perfusion with diazoxide.At 60 min of reperfusion,the hearts were sliced and stained for determination of myocardial infarct size (IS) as a percentage of area at risk (AAR) (IS/AAR),cell apoptosis and expression of phosphorylated STAT3 (p-STAT3) protein (by Western blot) and STAT3 mRNA (using RT-PCR).Apoptotic index (AI) was calculated.Results Compared with group C,the IS/AAR and AI were significantly increased in the other four groups,the expression of p-STAT3 and STAT3 mRNA was down-regulated in I/R and Stattic groups,and the expression of p-STAT3 was down-regulated and STAT3 mRNA was up-regulated in P and DZX groups (P ＜ 0.05).Compared with group I/R,the IS/AAR and AI were significantly decreased,and the expression of p-STAT3 and STAT3 mRNA was up-regulated in P and DZX groups (P ＜ 0.05),and no significant changes were found in the parameters mentioned above in Stattic group (P ＞ 0.05).The IS/AAR and AI were significantly lower,and the expression of p-STAT3 and STAT3 mRNA was higher in DZX group than in P group (P ＜ 0.05).Conclusion STAT3 signal transduction pathway is involved in diazoxide cardioplegic solution-induced reduction of I/R injury in isolated rat hearts. Key words: STAT3 transcription factor; Diazoxide; Myocardial reperfusion injury

Abstract 68: Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy.

Abstract Introductory sentence: Increased activity of signal transducer and activator of transcription 3 (STAT3) is common in human malignancies, including colorectal cancers. Recently, we reported that STAT3 expression correlated with resistance to 5-fluorouracil (5-FU) based chemoradiotherapy. This is of considerable clinical relevance, because a large proportion of rectal cancers are resistant to preoperative multimodal treatment. We therefore examined whether STAT3 contributes to resistance to chemoradiotherapy. Experimental procedures: STAT3 mRNA and protein expression levels were determined in 12 colorectal cancers cell lines. STAT3 was inhibited using two different siRNAs and a small-molecular inhibitor (STATTIC) in the cell lines SW480 and SW837. Successful RNAi-mediated silencing of STAT3 or inhibition of phosphoSTAT3(Tyr705) was detected by Western blot and reduction of transcription factor activity was measured by a luciferase reporter assay. Additionally, we established doxycycline-inducible stable shRNA single cell populations and a non-silencing shRNA (shNEG) in SW480. To test the influence of STAT3 knock down or inhibition, clonogenic survival assays were performed. Therefore, RNAi or inhibitor treated cells were exposed to chemoradiotherapy using 3μM 5-FU and X-ray-irradiation at 1, 2, 4, 6, and 8 Gy. Finally, we tested the effect of a chemoradiotherapy combined with STATTIC treatment in a SW837 xenograft model in NMRI nude mice. To verify the sensitizing effect of STATTIC, tumor growth was recorded and growth delay assays were performed. Data: STAT3 was overexpressed in resistant cells at mRNA and protein level. siRNA transfected SW480, SW837, and SW480shRNA single cell clones showed a significant reduction of STAT3 protein and transcription factor activity after 96 hours. STATTIC inhibition led to a decreased phosphorylation of STAT3 after 1 hour. The silencing/inhibition resulted in a significantly increased chemoradiosensitivity with dose-reduction factors of 1.3 to 2.5 at a surviving fraction of 0.37. In vivo, additional STAT3 inhibition during chemoradiotherapy led to a profound chemoradiosensitization effect and a significant tumor growth delay in STATTIC treated mice. Survival of these mice was also enhanced, if compared to the control group. Conclusions: STAT3 is highly overexpressed in resistant colorectal cancer cells, and silencing or inhibition of STAT3 leads to a significantly increased chemoradiosensitivity in vitro and in vivo. This highlights the potential relevance of STAT3 for mediating treatment resistance and provides a first proof of concept that STAT3 represents a novel molecular target in rectal cancer to sensitize a priori resistant colorectal tumor cells to chemoradiotherapy. Citation Format: Melanie Spitzner, Birte Roesler, Christian Bielfeld, Carolin Herzberg, Georg Emons, Jochen Gaedcke, Margret Rave-Fränk, Tim Beißbarth, Thomas Ried, B. Michael Ghadimi, Marian Grade. Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2013-68

Role of signal transducer and activator of transcription 3 signaling pathway in reduction of myocardial ischemia-reperfusion injury by diazoxide postconditioning in rats

Objective To evaluate the role of signal transducer and activator of transcription 3 (STAT3) signaling pathway in the reduction of myocardial ischemia-reperfusion (I/R) injury by diazoxide postconditioning in rats.Methods Sixty adult male Sprague-Dawley rats,aged 3 months,weighing 240-260 g,were randomly divided into 5 groups (n =12 each):sham operation group (S group),I/R group,vehicle group (V group),diazoxide postconditioning group (D group),and STAT3 signaling pathway inhibitor Stattic group (St group).Myocardial I/R was produced by 30 min occlusion of left anterior descending branch of coronary artery followed by 120 min reperfusion.In V and D groups,0.4％ dimethyl sulfoxide and 7 mg/kg diazoxide (in 1 ml of 0.4％ dimethyl sulfoxide) were injected through the femoral vein at the onset of reperfnsion,respetively.In St group,Stattic was injected through the femoral vein 10 min before reperfusion,and the other procedures were the same as those in D group.The infarct size (IS) and myocardial apoptosis were detected by TTC staining and TUNEL,respectively.Apoptotic index (AI) was calculated.STAT3 mRNA expression in myocardial tissues was detected using RT-PCR.Western blot was used to detect the phosphorylation of STAT3.Results Compared with S group,the IS and AI were significantly increased and the expression of STAT3 mRNA and phosphorylation of STAT3 were decreased in I/R group (P ＜ 0.05).Compared with I/R group,the IS and AI were significantly decreased and the expression of STAT3 mRNA and phosphorylation of STAT3 were increased in D group (P ＜ 0.05).There was no significant difference in IS,AI,expression of STAT3 mRNA and phosphorylation of STAT3 between V group and St group (P ＞0.05).Compared with group D,the IS and AI were significantly increased and the expression of STAT3 mRNA and phosphorylation of STAT3 were decreased in St group (P ＜ 0.05).Conclusion STAT3 signaling pathway is involved in the reduction of myocardial I/R injury by diazoxide postconditioning in rats. Key words: STAT3 transcription factor; Diazoxide ; Myocardial reperfusion injury; Apoptosis

Expression of STAT3 in non-small cell lung cancer and its clinical significance

Signal transducer and activator of transcription 3(STAT3) plays an important role in tumor occurrence and development. Here we attempt to evaluate the clinical diagnostic value of STAT3 as a potential tumor marker by detecting the expression of STAT3 in blood of non-small cell lung cancer (NSCLC) patients. Immunohistochemistry was applied to detect STAT3 protein in A549 cells and MRC-5 cell, real time PCR was applied to detect STAT3 mRNA and ELISA was applied to detect the expression of STAT3 protein in NSCLC patients, benign lung disease group and healthy controls. The expression levels of STAT3 mRNA and STAT3 protein in A549 cells and NSCLC patients were significantly higher (P P > 0.05). When comparing the Az of STAT3 protein with CEA, CA125 and CYFRA21-1 respectively, there was no significant difference (P > 0.05). And we compared the Az of the four joint detection (STAT3 + CEA + CA125 + CYFRA21-1) with the three joint detection (CEA + CA125 + CYFRA21-1), 0.930 and 0.841, respectively, P

Correlation of STAT3, CEA in lung adenocarcinoma cell A549

The purpose of this study was to analyze the relationship between signal transducer and activator of transcription 3 (STAT3) and carcinoembryonic antigen (CEA) in lung adenocarcinoma cell A549, and to explore the value of STAT3 on early diagnosis of lung adenocarcinoma. The expression of CEA, STAT3 mRNA and it’s protein in human lung adenocarcinoma cell A549 and normal human lung cells MRC-5 were tested by immunohistochemistry staining (PV) and quantitative real time fluorescent PCR. The correlation between STAT3 and CEA in human lung adenocarcinoma cell A549 was analyzed. The protein and mRNA levels of STAT3, CEA in lung adenocarcinoma cell A549 were apparently higher than those in normal human lung cells MRC-5. The levels of STAT3 mRNA and it’s protein were positively correlated with CEA in lung adenocarcinoma cell A549. STAT3 have the same value in diagnosis of lung adenocarcinoma.

Interleukin-6 promotes myogenic differentiation of mouse skeletal muscle cells: role of the STAT3 pathway

Myogenic differentiation of skeletal muscle cells is characterized by a sequence of events that include activation of signal transducer and activator of transcription 3 (STAT3) and enhanced expression of its target gene Socs3. Autocrine effects of IL-6 may contribute to the activation of the STAT3-Socs3 cascade and thus to myogenic differentiation. The importance of IL-6 and STAT3 for the differentiation process was studied in C2C12 cells and in primary mouse wild-type and IL-6(-/-) skeletal muscle cells. In differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA expression and protein secretion started after increased phosphorylation of STAT3 on tyrosine 705 and increased mRNA expression of Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in differentiating C2C12 myoblasts impaired the expression of the myogenic markers myogenin and MyHC IIb and subsequently myotube fusion. However, the knockdown of IL-6 did not prevent the induction of STAT3 tyrosine phosphorylation. The IL-6-independent activation of STAT3 was verified in differentiating primary IL-6(-/-) myoblasts. The phosphorylation of STAT3 and the expression levels of STAT3, Socs3, and myogenin during differentiation were comparable in the primary myoblasts independent of the genotype. However, IL-6(-/-) cells failed to induce MyHC IIb expression to the same level as in wild-type cells and showed reduced myotube formation. Supplementation of IL-6 could partially restore the fusion of IL-6(-/-) cells. These data demonstrate that IL-6 depletion during myogenic differentiation does not reduce the activation of the STAT3-Socs3 cascade, while IL-6 and STAT3 are both necessary to promote myotube fusion.

Regulation of Human Endometrial Stromal Proliferation and Differentiation by C/EBPβ Involves Cyclin E-cdk2 and STAT3

During each menstrual cycle, the human uterus undergoes a unique transformation, known as decidualization, which involves endometrial stromal proliferation and differentiation. During this process, the stromal cells are transformed into decidual cells, which produce factors that prepare the uterus for potential embryo implantation. We previously identified the transcription factor CCAAT/enhancer-binding protein (C/EBP)β as a regulator of endometrial stromal proliferation and differentiation in mice. In this study, we addressed the role of C/EBPβ in human endometrial decidualization. Using small interfering RNA targeted to C/EBPβ mRNA, we demonstrated that C/EBPβ controls the proliferation of primary human endometrial stromal cells (HESCs) by regulating the expression of several key cell cycle-regulatory factors during the G(1)-S phase transition. Additionally, loss of C/EBPβ expression blocked the differentiation of HESCs in response to estrogen, progesterone, and cyclic AMP. Gene expression profiling of normal and C/EBPβ-deficient HESCs revealed that the receptor for the cytokine IL-11 and its downstream signal transducer signal transducer and activator of transcription 3 (STAT3) are targets of regulation by C/EBPβ. Chromatin immunoprecipitation analysis indicated that C/EBPβ controls the expression of STAT3 gene by directly interacting with a distinct regulatory sequence in its 5'-flanking region. Attenuation of STAT3 mRNA expression in HESCs resulted in markedly reduced differentiation of these cells, indicating an important role for STAT3 in decidualization. Gene expression profiling, using STAT3-deficient HESCs, showed an extensive overlap of pathways downstream of STAT3 and C/EBPβ during stromal cell differentiation. Collectively, these findings revealed a novel functional link between C/EBPβ and STAT3 that is a critical regulator of endometrial differentiation in women.

Reduction of STAT3 expression induces mitochondrial dysfunction and autophagy in cardiac HL-1 cells

Signal transducer and activator of transcription 3 (STAT3) is an important mediator of cardiac survival pathways. Reduced levels of STAT3 in patients with end-stage heart failure suggest a clinical relevance of STAT3 deficiency for cardiac disease. The recent identification of STAT3 as a mitochondrial protein which is important for full activity of mitochondrial complex I has opened a new field for the investigation of how STAT3 functions in cardioprotection. The goal of this study was to establish a cell culture model with a reduced STAT3 expression, and to use this model for the investigation of mitochondrial and mitochondrial-associated functions under STAT3 deficiency. In the murine cardiomyogenic cell line HL-1, the expression of STAT3 was silenced by lentiviral transduction with anti-STAT3 shRNA (STAT3 KD cells). STAT3 mRNA and protein levels were significantly reduced in HL-1 STAT3 KD cells compared to HL-1 cells transduced with a control shRNA. Spectrophotometric and polarographic assays with mitochondrial enriched fractions and intact cells showed reduced activities of respiratory chain complexes I, II, III and IV in HL-1 STAT3 KD cells. At ultrastructural level, a severe damage of mitochondrial integrity was observed, combined with a significant increase in autophagolysosomes in STAT3-deficient HL-1 cells. Our results demonstrate that the HL-1 STAT3 KD cell line is a good model to study cellular consequences of STAT3 deficiency. Moreover, this is the first study to show that STAT3 deficiency leads to a disruption of mitochondrial ultrastructure and increased autophagy.

Expression of interferon-γ, interferon-α and related genes in individuals with Down syndrome and periodontitis

BackgroundRecently, attenuation of anti-inflammatory and increase of pro-inflammatory mediators was demonstrated in individuals with Down syndrome (DS) in comparison with euploid patients during periodontal disease (PD), suggesting a shift to a more aggressive inflammation in DS. AimTo determine the influence of DS in the modulation of interferons (IFNs) signaling pathway in PD. Materials and methodsClinical periodontal assessment was performed and gingival tissue samples obtained from a total of 51 subjects, including 19 DS individuals with PD, 20 euploid individuals with PD and 12 euploid individuals without PD. Expression levels of interferon-gamma (IFNG) and interferon-alpha (IFNA), and their receptors IFNGR1, IFNGR2, IFNAR1 and IFNAR2, the signaling intermediates Janus kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1) were determined using real time quantitative polymerase chain reaction (qPCR). ResultsClinical signs of periodontal disease were markedly more severe in DS and euploid patients with PD in comparison to euploid and periodontally healthy patients. There was no difference on mRNA levels of IFNA, IFNG, INFGR2, IFNAR1 and IFNAR2 between DS and euploid individuals, even though some of these genes are located on chromosome 21. STAT1 and IRF1 mRNA levels were significantly lower in DS patients in comparison with euploid individuals with PD. In euploid individuals, PD was associated with an increased expression of IFNGR1, IFNGR2, IFNAR1, STAT1 and IRF1. ConclusionsReduced expression of STAT1 and IRF1 genes indicate an impaired activation of IFNs signaling in individuals with DS and PD. Expression of IFNA, IFNG and IFN receptors was not altered in DS patients, indicating that indirect mechanisms are involved in the reduced activation of IFN signaling.

Expression of the Interleukin‐10 Signaling Pathway Genes in Individuals With Down Syndrome and Periodontitis

Individuals with Down syndrome (DS) have a higher prevalence and severity of periodontal disease, which cannot be explained by poor oral hygiene alone and is related to changes in the immune response. The aim of this study is to evaluate whether DS was associated with differential modulation of expression of genes associated with proinflammatory and anti-inflammatory responses in periodontal disease. A total of 51 individuals were evaluated: 19 individuals with DS and periodontal disease (group 1), 20 euploid individuals with periodontal disease (group 2; positive control), and 12 euploid individuals without periodontal disease (group 3; negative control). Clinical periodontal evaluation and gingival biopsies were performed. Quantitative reverse transcription-polymerase chain reaction was used to determine expression levels of interleukin-10 (IL-10), the receptors IL-10RA and IL-10RB, intracellular adhesion molecule 1 (ICAM-1), interferon-γ-inducible protein 10 (IP-10), and the signaling intermediates Janus kinase 1, signal transducer and activator of transcription 3 (STAT-3), and suppressor of cytokine signaling 3 (SOCS-3). Expression of IL10, SOCS3, IP10, and ICAM1 mRNA in DS patients was significantly lower compared to euploid individuals with periodontal disease, whereas IL-10RB and STAT-3 mRNA levels were higher in individuals with DS. Reduced expression of IL-10 coupled with a possible increase of STAT3 activation (increase of STAT3 and reduction of SOCS3 mRNA) indicates an important modulation of the immune response, with attenuation of anti-inflammatory and increase of proinflammatory mediators. This modulation may be related to the increased prevalence and severity of periodontitis in individuals with DS.

Abstract 3446: Stat3 is a potential molecular target for chemoradiosensitization of colorectal cancer cells

Abstract Introductory sentence: Resistance to preoperative chemoradiotherapy represents a major clinical problem in the treatment of patients with locally advanced rectal cancer. Therefore, the identification of novel molecular targets that are differently expressed in responsive and resistant tumors is exceedingly important. Furthermore, modulating target gene expression may sensitize a priori resistant tumors to multimodal therapy. Experimental procedures: Previously, we established an in vitro model for chemoradiotherapy in 12 colorectal cancer cell lines, and correlated differences in treatment sensitivity with pretherapeutic gene expression profiles. Within our signature we identified the signal transducer and activator of transcription 3 (STAT3), which was significantly correlated with treatment resistance. To test the functional relevance of the observed over-expression of STAT3, we first determined STAT3 mRNA and protein expression levels in all 12 cell lines. Subsequently, we established doxycycline-inducible stable shRNA single cell populations and a non-silencing shRNA (shNEG) in SW480. The induced single cell clones and shNEG were treated with 3µM 5-FU and exposed to 1, 2, 4, 6, and 8 Gy of X-rays. In addition, STAT3 was inhibited using two different siRNAs and a small-molecular inhibitor (STATTIC). Successful RNAi-mediated silencing of STAT3 and inhibitor treatment was detected by Western blot. Data: STAT3 was significantly overexpressed in resistant cells at mRNA and protein level. In stable STAT3 knockdown single cell clones and transiently siRNA transfected SW480 and SW837 cells, successful silencing of the STAT3 protein was detected after 72 hours, and STATTIC-induced inhibition of STAT3-phosphorylation after 1 hour. The silencing/inhibition resulted in a significantly increased chemoradiosensitivity with dose-reduction factors of 1.8 to 2. Conclusions: STAT3 is highly overexpressed in resistant colorectal cancer cells, and silencing of STAT3 leads to a significantly increased chemoradiosensitivity. This highlights the potential relevance of STAT3 as a novel molecular target in rectal cancer to sensitize a priori resistant colorectal tumor cells to chemoradiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3446. doi:1538-7445.AM2012-3446

The efficiency of silencing expression of the gene coding STAT3 transcriptional factor and susceptibility of bladder cancer cells to apoptosis

Aim of the studyAbnormalities in signaling as well as altered gene expression have been identified in numerous diseases, including cancer. The biological functions of signal transducer and activator of transcription 3 (STAT3) are very broad. It is thought that STAT3 can also contribute to oncogenesis. RNA interference (RNAi) is one of the most efficient tools for silencing gene expression within cells. The main goal of the study was to verify the effectiveness of STAT3 gene silencing and its influence on cell proliferation and activation of apoptosis in bladder cancer cells.Material and methodsThe study was conducted on cellular material, which was the stable human bladder cancer cell line T24. The synthesis of shRNA (short hairpin RNA) interfering with the STAT3 gene was based on pSUPER. neo expression vector. The gene expression at the mRNA level was determined by the real-time PCR method. The influence of STAT3 gene silencing on apoptosis induced in cells with modulated STAT3 expression was evaluated using parallel quantification of mono- and oligonucleosomal DNA degradation of genomic DNA.ResultsIn transfected T24 cells, the STAT3 mRNA expression decreased to the level of 68.3% compared to the scrambled (SCR) control. Silencing the STAT3 gene induced changes in the phenotype of T24 cells. Statistically significant differences in cell proliferation (p = 0.0318) and apoptosis induction (p = 0.0376) were observed.ConclusionsApplication of the designed shRNA for the STAT3 gene contributed to a decrease of expression of the examined gene. It also decreased the proliferation and increased the susceptibility to apoptosis in T24 bladder cancer cells.

Prognostic significance of phosphorylated signal transducer and activator of transcription 3 and suppressor of cytokine signaling 3 expression in human cutaneous melanoma

Cutaneous malignant melanoma is one of the most common and aggressive forms of human cancers and has a poor prognosis. Activation of signal transducer and activator of transcription 3 (STAT3) has been found in several human cancers and is thought to correlate aggressive disease and poor response. In this study, we investigated the clinical role of STAT3 and its natural inhibitor, suppressor of cytokine signaling 3 (SOCS3), in human cutaneous melanoma development and progression. Immunohistochemical analysis of pSTAT3, SOCS3, matrix metalloproteinase (MMP)-2, and MMP-9 expression was performed on 90 primary melanomas and 43 common melanocytic nevi specimens. The expression of STAT3 mRNA was further detected by in-situ hybridization in the same cohort of patients. The association of STAT3 mRNA, pSTAT3, and SOCS3 protein expression with clinicopathological parameters and patient survival was analyzed. Altered expression of STAT3 mRNA, pSTAT3, and SOCS3 protein was observed in melanoma specimens, compared with benign melanocytic nevi. High expression of pSTAT3 was correlated to large tumor diameter, depth of tumor invasion, tumor lymph node metastasis, MMP-2 and MMP-9 expression, and poor patient survival. Decreased expression of SOCS3 was correlated to depth of tumor invasion, tumor lymph node metastasis, the expression of MMP-2, MMP-9, and pSTAT3, and poor patient survival. Moreover, the expression of pSTAT3 was conversely correlated to SOCS3 expression in melanoma. Our results indicate that deregulated expression of pSTAT3 and SOCS3 might possess potential roles in the development and progression of human cutaneous melanoma.