mRNA Expression Levels Of STAT3 Research Articles

CHRNA9 as a New Prognostic Marker and Potential Therapeutic Target in Glioma.

Background: The nicotinic acetylcholine receptor (nAChR) subunit alpha-9 (CHRNA9) is a unique cholinergic receptor, which is involved in tumor proliferation, apoptosis, metastasis and chemotherapy resistance. However, the correlation between the expression level of CHRNA9 in glioma and the clinical features and prognosis of glioma patients has not been clarified. The aim of this study was to verify the expression level of CHRNA9 in glioma and its effect on prognosis by bioinformatics methods. Methods: The RNA-seq data of glioma and normal samples were obtained from the TCGA and GTEx databases. Bioinformatics methods were utilized to analyze the differential expression of CHRNA9 between tumor samples and normal samples. The potential association between CHRNA9 and the clinicopathological features of glioma patients was also investigated. The Kaplan-Meier method and Cox regression were utilized to analyze the relationship between CHRNA9 expression level and survival time and prognostic value of glioma patients. Enrichment analysis was applied to predict gene function and signaling pathways associated with CHRNA9. Experimental verification was performed using tumor tissues and paracancerous tissues from glioma patients. Results: The results of bioinformatics analysis showed that the expression of CHRNA9 was increased in glioma tissues, correlating with poor prognosis and reduced patient survival time. Enrichment analysis suggested that CHRNA9 may interact with the JAK/STAT pathway. CHRNA9 was also found to be abnormally expressed in various other tumors and associated with the expression levels of numerous immune checkpoints in glioma. The findings from the analysis of clinical samples revealed that the expression levels of both mRNA and protein of CHRNA9 in glioma tissues were higher than those in paracancerous tissues. Similarly, the mRNA expression levels of STAT3, IL-6, and TNF-α, which are crucial factors in the STAT3 pathway, were elevated in glioma tissues compared to paracancerous tissues. Conclusion: CHRNA9 is a potential prognostic marker and immunotherapy target for glioma, with its mechanism of action potentially linked to the STAT3 pathway.

Crosstalk between heat shock factor 1 and signal transducer and activator of transcription 3 mediated by interleukin-8 autocrine signaling maintains the cancer stem cell phenotype in liver cancer.

Liver cancer stem cells (LCSCs) cause therapeutic refractoriness and relapse in hepatocellular carcinoma. Heat shock factor 1 (HSF1) plays versatile roles in multiple cancers. However, the role of HSF1 in LCSCs is not well understood. This study investigated the function and signal mechanisms of HSF1 in maintaining LCSC phenotypes. We established two LCSC lines, HepG2-R and HuH-7-R. Constitutive activation of HSF1 was observed in these LCSCs. Specific short hairpin RNAs (shRNAs) and chemical inhibitors were used to identify the relationship between HSF1 expression and LCSCs phenotypes. We revealed a concomitant activation modality involving HSF1 and STAT3 in LCSCs and liver cancer tissues. We also found that liver cancer patients whose HSF1 and STAT3 mRNA expression levels were high presented with unfavorable clinicopathological characteristics. Moreover, the secretion of interleukin-8 (IL-8) was elevated in the LCSC medium and was directly regulated by HSF1 at the transcriptional level. In turn, IL-8 activated HSF1 and STAT3 signaling, and a neutralizing IL-8 antibody inhibited HSF1 and STAT3 activity, reduced cancer stem cell marker expression, and decreased LCSC microsphere formation. Simultaneous intervention with HSF1 and STAT3 led to synergistically suppressed stemness acquisition and growth suppression in the LCSCs in vivo and in vitro. Our study indicates that IL-8 mediates the crosstalk between the HSF1 and Stat3 signaling pathways in LCSCs and that the combined targeting of HSF1 and STAT3 is a promising treatment strategy for patients with advanced liver cancer.

Effects of miR-124-3p Silencing on Neuronal Damage in the Hippocampus of Depression Rats by Regulating STAT3 Gene.

Objective A large amount of evidence shows that the abnormal expression of miRNA plays an important role in the development of depression. Therefore, we investigated the effect of miR-124-3p on neuronal damage in the hippocampus of depression rats. Methods The target genes of miR-124-3p were predicted by the database; the depression model was prepared by subcutaneous injection of corticosterone (CORT), and LV-miR-124-3p asponge lentiviral suspension was given to determine the weight of rats and open-field test, sugar preference experiment, Serum CORT, 5-HT, DA, and NE were measured, observe and record the behavior of rats, including behavior, diet, and hair. The expression of miR-124-3p, STAT3, Bcl-2, and Bax in rat hippocampus was measured. The rat hippocampal neuron cells were extracted and transfected with miR-124-3p inhibitor; the cells were cultured with CORT, and the cell survival rate was evaluated by MTT experiment, and the expressions of miR-124-3p, STAT3, Bcl-2, and Bax in the cells were detected. Luciferase reporter gene verifies the targeted regulation of miR-124-3p on STAT3. Results Compared with depression rats, silencing miR-124-3p increased the weight of the rats, increased the number of open-field activities, and significantly improved the general state and pathological state of the rats. The sugar water preference rate was significantly increased, the CORT content in the serum of rats decreased significantly, and the levels of 5-HT, DA, and NE increased significantly. After the treatment of silencing miR-124-3p, the expression level of miR-124-3p was decreased, while the STAT3 mRNA and protein expression levels were increased. And the protein and mRNA expression levels of Bcl-2 were increased, and the Bax protein and mRNA expression were decreased. Cell experiments verified that silencing miR-124-3p increased cell survival, the expression level of miR-124-3p decreased remarkably, while the expression levels of STAT3 mRNA and protein increased significantly. Silencing miR-124-3p reversed the effects of CORT treatment on miR-124-3p and STAT3 in neuronal cells. The luciferase reporter gene experiment confirmed that miR-124-3p targets and regulates STAT3 expression. Conclusion Silencing miR-124-3p may protect hippocampal neurons from damage in depression rats by upregulating STAT3 gene.

Mechanism of TRIM27 promoting inflammatory response in lung cancer cells

Objective: To investigate the mechanism of tripartite motif-containing 27 (TRIM27) expression promoting inflammatory response in non-small lung cancer cells. Methods: Ten cases of lung cancer tissues and their matched normal tissue (the distance was 5 cm of the tumor marginal) from patients underwent resection in the People's Hospital of Pingyang Hospital Affiliated to Wenzhou Medical University were collected. The expression of TRIM27 was identified by using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. TRIM27 knockdown experiment included negative control (NC(TRIM27)) group, TRIM27 short interfering RNA (siRNA) group, NC(TRIM27)+ TNF-α group and TRIM27 siRNA+ TNF-α group. Interlukin-6 (IL-6) knockdown experiment included NC(IL-6) group and IL-6 siRNA group. The protein expressions of TRIM27, TNFR1, TNFR2 and some TNFR related inflammation factors were verified by qRT-PCR and WB. Results: The expression levels of TRIM27 in NSCLC tissues of different stages (stage Ⅰ: 2.81±0.58, stage Ⅱ: 3.32±1.38, stage Ⅲ: 3.67±1.24) was higher than that in the adjacent normal tissues (1.01±0.15, 0.92±0.10 and 1.05±0.12, P<0.05). The expression levels of TRIM27 mRNA in NC(TRIM27) group and NC(TRIM27)+ TNF-α group were 0.94±0.12 and 1.67±0.03, and the expression levels of TRIM27 protein were 0.31±0.02 and 0.38±0.01, respectively (P<0.05). The expression levels of IL-6 mRNA in NC(TRIM27)+ TNF-α group and TRIM27 siRNA+ TNF-α group were 11.35±0.12 and 5.62±0.15, respectively, and the expression levels of VCAM-1 mRNA were 18.75±0.17 and 9.35±0.11, respectively. STAT3 mRNA expression levels were 16.54±0.10 and 8.12±0.10, respectively, with statistical significance (P<0.05). The expression levels of IL-6 mRNA in NC(IL-6) group and IL-6 siRNA group were 1.10±0.07 and 0.52±0.16, respectively, and the expression levels of STAT3 mRNA were 1.01±0.01 and 0.48±0.12, respectively. The expression levels of TRIM27 mRNA were 1.03±0.01 and 0.30±0.11, respectively, with statistical significance (P<0.05). Conclusion: The upregulation of TRIM27 in NSCLC tissue and cells promotes the expression of TNF-α, and may activate inflammatory response by regulating TNF-α-induced IL-6/STAT3 signaling pathway.

Relationship between miR-338-3p and Clinicopathological Parameters, Prognosis, and STAT3 mRNA Expression in Nasopharyngeal Carcinoma.

Objective To investigate the expression of miR-338-3p in nasopharyngeal carcinoma (NPC) and its relationship with STAT3 mRNA expression as well as their relationship with clinical pathological parameters and prognosis of patients. Methods From September 2016 to September 2018, 71 patients with NPC were selected as the NPC group, and 71 samples of NPC tissues were collected during the operation. A total of 23 patients who underwent biopsy due to chronic nasopharyngitis were selected as the control group and 23 nasopharyngeal mucosal tissues were collected. The expressions of miR-338-3p and STAT3 mRNA in nasopharyngeal tissue of two groups were detected by real-time quantitative PCR, and the relationship between the two was analyzed. To collect clinical data of NPC patients and analyze the relationship between the expressions of miR-338-3p and STAT3 in NPC tissues and clinical pathological parameters of the patients, we followed up the patients with nasopharyngeal carcinoma for three years to observe the relationship between miR-338-3p, STAT3, and the prognosis of the patients. Results The relative expression levels of miR-338-3p in nasopharyngeal tissues of the NPC group and the control group were 0.39 ± 0.05 and 1.01 ± 0.09, respectively (P < 0.05). The relative expression levels of STAT3 mRNA in nasopharyngeal tissues of the NPC group and the control group were 3.82 ± 0.21 and 1.04 ± 0.11, respectively (P > 0.05). miR-338-3p was negatively correlated with the relative expression of STAT3 mRNA in nasopharyngeal carcinoma (r = 0.038, P > 0.05). The expression of miR-338-3p was related to the age of the patient, clinical TNM stage, T stage, and distant metastasis (all P < 0.05). STAT3 expression was correlated with clinical TNM stage, T stage, and distant metastasis in our patient (P < 0.05). The expressions of miR-338-3p and STAT3 in nasopharyngeal carcinoma tissues from different gender, histological type, N stage, M stage, and degree of differentiation showed no statistical differences (P > 0.05). The survival rate of the group with low miR-338-3p expression was significantly lower than that of the group with high miR-338-3p expression (P > 0.05). The survival rate of patients with the high STAT3 expression group was significantly lower than that of patients with the low STAT3 expression group (P > 0.05). Conclusion There is a negative correlation between the low expression of miR-338-3p and the high expression of STAT3 in NPC, which are all related to the TNM stage, T stage, and prognosis of the patient.

Escalated activation ofSTAT3 due to JAK2 V617F activatingmutation causesdownregulation ofPTEN via miRNA 21overexpression

Abstract Background Mutations in Janus kinase 2 (JAK2) genes are the genetic characteristic of BCR-ABL1-negative myeloproliferative neoplasms (MPN). JAK2 V617F mutation is frequently found in Polycythemia Vera (PV) and Essential Thrombocythemia (ET). We aimed to determine the effect of JAK2 V617F on expression of phosphatase and tensin homolog (PTEN) and its regulator miR-21. Materials and Methods Seventy two (72) (MPN) patients were screened for JAK2 V617F burden using ASO-PCR. Out of 72 MPN patients, 58 were JAK2 V617F positive and 14 were JAK2 V617F negative. BCR/ABL1 fusion gene transcript screening by multiplex PCR was done to rule out Philadelphia positive MPN. We performed SYBR green based qRT-PCR assay to characterize relative STAT3, PTEN mRNA expression and miR21 expression in 72 MPN patients. Results The mRNA expression levels of STAT3 and PTEN and mi-21 expression in all MPN patients were evaluated and compared withJAK2 (V617F) and JAK2 (WT) MPN patients. We identified that STAT3 mRNA expression was significantly upregulated in patients with JAK2 (V617F) genotype (mean fold±SD=2.14 ±1.2) compared to patients with JAK2 (WT) genotype (mean fold±SD=1.23±0.95). Further, we observed a significantly increased miR 21 expression (mean fold±SD=2.18±0.44) in JAK2 (V617F) patients in comparison to JAK2 (WT) patients (mean fold±SD=1.70±0.49) (p = 0.0003). In addition, we observed that PTEN expression was substantially and significantly downregulated in JAK2 (V617F) patients (mean fold±SD= 1.73 ± 0.36) compared JAK2 (WT) patients (mean fold±SD=2.55 ± 0.25) (p Conclusion In summary, STAT3 activation caused by JAK (V617F) mutation in MPN patients causes overexpression of miR-21 which leads to downregulation of tumour supressor PTEN MPN patients. Thus, our suggests that JAK2 activated STAT3/miRNA-21 may play a valuable prognostic biomarker for MPN.

Antitumor effects of saikosaponin b2 on breast cancer cell proliferation and migration.

Saikosaponin b2 (SSb2) can be extracted from Bupleurum spp. roots (Radix Bupleuri), which belongs to the Umbelliferae family. The current study aimed to explore the effects of SSb2 on proliferation of breast cancer cells and to identify the mechanism by which SSb2 affects breast cancer cell migration. mRNA expression levels of STAT3 and vasodilator‑stimulated phosphoprotein (VASP) were determined and increased expression was observed in 16 breast cancer tissues compared with the paracancerous tissues. MTT, wound healing, colony formation assays and western blot suggested that SSb2 inhibited MCF‑7 proliferation and migration. It was further identified by western blot analysis that SSb2 treatment reduced levels of phosphorylated STAT3, VASP, matrix metallopeptidase (MMP) 2 and MMP9 in MCF‑7 compared with the untreated cells. In addition, it was demonstrated that inhibition of STAT3 phosphorylation decreased VASP expression levels and induction of STAT3 phosphorylation increased VASP levels. Furthermore, it was observed that the treatment of Kunming mice with SSb2 at 30mg/kg/day for 30days induced no obvious changes in the liver or kidney tissues, as determined by haematoxylin and eosin staining. In conclusion, these results indicated that SSb2 may be a potential antitumor drug for the treatment of breast cancer, which acts by suppressing proliferation and migration by downregulating the STAT3 signalling pathway and inhibiting the expression of VASP, MMP2 and MMP9 expression.

Analysis of STAT1, STAT2 and STAT3 mRNA expression levels in the blood of patients with multiple sclerosis.

Multiple sclerosis (MS) is the most common chronic, inflammatory, autoimmune disease of the central nervous system (CNS) maintained by the secretion of a large number of cytokines [1]. The signal transducer and activator of transcription (STAT) family has an essential role in transmitting many of the cytokine-mediated signals and failure in the signaling process contributes to the etiopathogenesis of MS. This study aimed to assess STAT1, STAT2 and STAT3 gene expression in the blood of 50 relapsing-remitting MS (RR-MS) patients and 50 healthy controls by TaqMan Quantitative Real-Time PCR. The results showed that STAT1 gene expression was significantly up-regulated (p= 0.023), whereas STAT2 gene expression was significantly down-regulated (p< 0.0001) in MS patients compared to controls. On the other hand, there was no significant difference between MS patients and controls for STAT3 gene expression (p= 0.837). In addition, there was no significant correlation between the expression of STAT1, STAT2, STAT3 genes and clinical findings, such as the level of physical disability in MS patients (according to the Kurtzke Expanded Disability Status Scale (EDSS) criterion) and disease duration. A significant positive correlation was demonstrated between STAT1 and STAT2 and also between STAT1 and STAT3. This study shows for the first time that a comparison of the relative quantitative expression of three different STAT genes in the blood cells of MS patients compared to controls revealed marked differences in the expression of the STAT family genes that might reflect their different roles in the pathogenesis of MS. These transcripts might be useful biomarkers for evaluating the efficacy of IFN treatment of the MS patients.

Effects of Rituximab on JAK-STAT and NF-κB signaling pathways in acute lymphoblastic leukemia and chronic lymphocytic leukemia

Abstract Objectives Rituximab is a monoclonal antibody that targets the B-lymphocyte surface antigen CD20. It is used in the treatment of some diseases including B-cell chronic lymphocytic leukemia (B-CLL). There are a lot of data regarding effect of Rituximab on lymphoma cells. But, there is no satisfactory information about the effect of Rituximab on the signaling pathways in leukemia cells. In this study, it was aimed to understand the effect of Rituximab on JAK-STAT and NF-κB signaling pathways in B-cell acute lymphoblastic leukemia (B-ALL) and B-CLL. Material and methods Apoptotic effect of Rituximab in the TANOUE (B-ALL) and EHEB (B-CLL) cell lines were evaluated by using the Annexin V method. mRNA expression levels of STAT3 and RelA were analysed by quantitative RT-PCR (Q-PCR). Alterations in STAT3 and RelA protein expressions were detected by using a chromogenic alkaline phosphatase assay after Western Blotting. Results Rituximab had no apoptotic effect on both cell lines. Complement-mediated cytotoxicity was only detected in EHEB cells. mRNA and protein expressions of STAT3 and RelA genes were decreased following Rituximab treatment. Conclusion Our preliminary results suggest that the use of Rituximab might be effective in B-ALL though both signaling pathways.

Effect of the JAK2/STAT3 signaling pathway on nerve cell apoptosis in rats with white matter injury.

The Janus activated kinase 2 (JAK2)/signal transducer and the activator of transcription 3 (STAT3) pathway are involved in many physiological processes, such as cell survival, inflammation, development, proliferation and differentiation. Increasing evidence has shown that this pathway also has neuron-specific functions in the central nervous system. In this study, the functional significance of the JAK2/STAT3 signaling pathway in nerve cell apoptosis in rats with white matter injury was evaluated. The rat model of white matter injury was established by ligating bilateral common carotid arteries, and the changes of the JAK2 and STAT3 phosphorylation in hippocampal neurons were evaluated using the immunohistochemistry. In addition, the effects of JAK2 inhibitor AG490 and STAT3 small interfering ribonucleic acids (siRNAs) on the expression of phosphorylated-JAK2 (pJAK2), STAT3 messenger RNAs (mRNAs) and pSTAT3 in hippocampal neurons of white matter injury rats were studied. The effects of both on cerebral infarction volume and neuron apoptosis in white matter injury rats were also investigated. The expression of pJAK2 and pSTAT3 were significantly increased after white matter injury in rats (p<0.05). JAK2 inhibitor AG490 markedly decreased the phosphorylation of JAK2 and STAT3 in hippocampal neurons in the model group (p<0.05). STAT3 siRNAs remarkably reduced the expression levels of STAT3 mRNA and protein in hippocampus neurons in the model group (p<0.05), while having no effect on the expression level of pJAK2 protein. AG490 and STAT3 siRNAs notably attenuated the volume of cerebral infarction in the model group, as well as reduced neuron apoptosis after white matter injury. The inhibition of the JAK2/STAT3 signaling pathway contributed to reducing the volume of cerebral infarction and neuron apoptosis in rats with white matter injury.

Proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes following signal transducer and activator of transcription 3 RNA interference delivery.

In this study, we investigated the effects of delivering small interfering RNA (siRNA) for efficient STAT3 downregulation on propagation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). The FLSs were transfected with three different siRNAs. RNAi-1 was selected for further experiments. The expression levels of both STAT3 messenger RNA (mRNA) and its protein were detected by a real-time polymerase chain reaction and Western blot analysis. The proliferation of FLSs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The apoptosis of FLSs was examined by flow cytometry. The expression levels of cell apoptotic-related genes Bcl-2, Bax, and caspase-3 were detected by Western blot analysis. RNAi-1 was selected as the RNAi group for its lowest expression levels of STAT3 mRNA. In RNAi group, the proliferation of synoviocytes was much lower and the apoptosis rate was significantly higher. FLSs of RNAi-1 group showed significantly lower expression level of apoptotic-inhibiting gene Bcl-2 and significantly higher expression levels of proapoptotic gene Bax and apoptotic protease caspase-3. Transfection with targeted STAT3 recombinant plasmids effectively inhibited the expression of STAT3 mRNA and its protein in RA-FLSs. RNAi-mediated silencing of STAT3 reduced the proliferation and promoted the apoptosis of FLSs.

Abstract LB-396: Bile reflux-induced APE1 mediates activation of EGFR-STAT3 in barret's and esophageal adenocarcinoma cells

Abstract Background: Barrett's esophagus (BE) is a premalignant condition where columnar metaplasia replaces the stratified squamous epithelium of the distal esophagus and is regarded as the main risk factor for esophageal adenocarcinoma (EAC). BE develops as a consequence of chronic gastroesophageal reflux disease (GERD), where acid and bile salts abnormally refluxate from the stomach into the distal region of esophagus. APE1/REF-1 is a multifunctional protein that plays an essential role in DNA repair activity and in redox-dependent transcriptional regulation. STAT3 signaling has been implicated in regulating cell survival, angiogenesis, and metastasis. EGFR is well known to facilitate the tyrosine phosphorylation of STAT3 and initiates downstream signaling cascades that facilitate proliferation, tumorigenesis, metastasis, and chemo resistance. In this study, we investigated the role of APE1 in activation of STAT3 in response to acidic bile salts (ABS) in EAC. Methodology: Non-neoplastic Barrett's (CPA, BAR10T and CPB) and esophageal adenocarcinoma (OE33, FLO-1) cells were exposed to acidic bile salts (pH4.0 and 5.5, 100 and 200uM) for 20 minutes. The mRNA and protein expression levels of APE-1, STAT3 and target genes were evaluated by real time PCR and immunoblotting. STAT3 transcription activity was studied by luciferase reporter assay. Immunofluorescence and cytoplasmic-nuclear fractionation were used to examine localization of APE-1, EGFR and STAT3 after exposure to acidic bile salts. Results: We demonstrate that APE1 is constitutively overexpressed in EAC whereas its expression is induced in non-neoplastic BE in response to ABS. Using overexpression and shRNA knockdown of APE1, we found that APE1 is required for phosphorylation, nuclear localization, and transcription activation of STAT3. By using APE1 redox-specific mutant (C65A) and APE1 redox inhibitor (E3330), we demonstrate that APE1 activates STAT3 in a redox-dependent manner. By using pharmacologic inhibitors and genetic knockdown systems, we found that EGFR is a required link between APE1 and STAT3. Phosphorylation of EGFR (Y1068) was directly associated with APE1 levels and redox function. By using co-immunoprecipitation and proximity ligation assays, we found that APE1 coexists and interacts with the EGFR-STAT3 protein complex. Consistent with these findings, we demonstrated a significant induction in the mRNA expression levels of STAT3 target genes (IL-6, IL-17A, BCL-xL, Survivin and c-Myc) in BE and EAC cells, following ABS treatment. ChIP assays indicated that ABS-treatment enhances binding of STAT3 to the promoter of its target genes, Survivin and BCL-xL, following ABS treatment. Inhibition of APE1/REF-1 redox activity using E3330 abrogated STAT3 DNA binding and transcriptional activity. In summary, our results provide evidence that bile reflux induced APE1 is involved in the activation of EGFR-STAT3 signaling axis in BE and EAC. Conclusion: The redox activity of APE1 mediates EGFR- STAT3 activation as a survival mechanism in response to acidic bile salts and oxidative stress. Constitutive activation of APE1 in EAC may present a therapeutic opportunity to targeting redox vulnerabilities in cancer cells. Citation Format: Ajaz A. Bhat, Heng Lu, Mohammad Suotto, Chen Zheng, Dunfa Peng, Mohammad Haris, Abbes Belkhiri, Wael El-Rifai. Bile reflux-induced APE1 mediates activation of EGFR-STAT3 in barret's and esophageal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-396.

Resveratrol attenuates pro-inflammatory cytokines and activation of JAK1-STAT3 in BTBR T+ Itpr3tf/J autistic mice

Autism is a neurodevelopmental disorder characterized by qualitative impairment in communication, social interaction, and repetitive stereotypic behavior. Resveratrol plays a role in several disorders such as neuroimmune, autoimmune, and allergic disorders. BTBR T+ Itpr3tf/J (BTBR) mice, a model for autism, show several behavioral deficits that are physiological characteristics similar to those observed in patients with autism. Previous studies have shown that JAK-STAT signaling pathway is associated with many neurodevelopmental disorders. We investigated the possible role of resveratrol on IL-6+, TNF-α+, IFN-γ+, and STAT3+ in CD4+ T spleen cells in BTBR mice as compared to C57BL/6J mice. We also assessed the effect of resveratrol treatment on IL-6, TNF-α, IFN-γ, JAK1, and STAT3 mRNA expression levels in the brain tissue. We further assessed IL-6, IFN-γ, TNF-α, phosphorylated (p) JAK1, and pSTAT3 (Tyr705) protein expression levels in the brain tissue. Resveratrol (20 and 40 mg/kg)-treated mice had significantly decreased in IL-6+, TNF-α+, IFN-γ+, and STAT3+ in CD4+ spleen cells as compared with BTBR control mice. Resveratrol treatment also decreased IL-6, TNF-α, IFN-γ, JAK1, and STAT3 mRNA expression levels as compared with BTBR control mice in the brain tissue. Moreover, resveratrol treatment resulted in decreased protein expression levels of IL-6, IFN-γ, TNF-α, pJAK1, and pSTAT3 (Tyr705) as compared with BTBR control mice in the brain tissues. Taken together, these results indicate the efficacy of resveratrol in reducing cytokines and JAK-1/STAT3 signaling in BTBR mice, which is a novel and important finding and might be important for future therapies in neuroimmune dysfunction.

Correlation of IL-6R/STAT3 signaling with invasiveness of pituitary null cell adenomas: A study based on proteomics

Objective To identify the relationship of IL-6R/STAT3 signaling to invasiveness of pituitary null cell adenomas by the study of proteomics. Methods In this study, specimens were collected from 45 cases of pituitary null cell adenomas which were surgical treated at Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University. Normal pituitary tissues were collected from 3 body donors who died from traffic accidents. Different proteins between invasive and non-invasive null cell adenomas were determined by nano liquid chromatography tandem mass spectrometry (nano LC-MS/MS) and quantitative proteomics analysis. The signaling pathway related to invasive of null cell adenomas was identified by bioinformatic analysis. The gene expression levels of the signaling pathway was confirmed by qPCR (quantitative polymerase chain reaction) and immunohistochemistry (IHC) in invasive pituitary tumors and non-invasive null cell adenomas. Results A total of 283 differentially expressed proteins (P<0.05) were identified between invasive and non-invasive pituitary null cell adenomas. Among those, 182 and 101 were up-regulated and down-regulated in invasive pituitary null cell adenomas, respectively. The bioinformatic analysis demonstrated that 8 canonical pathways were differentially expressed between normal pituitary glands and pituitary null cell adenomas. IL-6R/STAT3 signaling was related to invasiveness of pituitary null cell adenomas. The IL-6R, JAK2 and STAT3 mRNA expression levels in invasive null cell adenomas (2.2, 2.8 and 4.3, respectively) were significantly higher than those in non-invasive null cell adenomas (1.7, 1.7, 2.9, respectively) based on the results of qPCR (P<0.05). The values of staining intensity of IL-6R, JAK2, STAT3, p-STAT3 and MMP9 (9.2, 8.6, 9.7, 10.5 and 9.8, respectively) were significantly increased in invasive null cell adenomas compared with those in non-invasive null cell adenomas (5.8, 5.3, 5.7, 5.2 and 4.1, respectively) according to the IHC data. Conclusion The IL-6R/STAT3 signaling may play an important role in invasive null cell adenomas by enhancement of MMP9 expression. Key words: Pituitary adenoma; Neoplasm invasiveness; Proteomics; Signal pathways; IL-6R/STAT3

Inhibition of prostate cancer RM1 cell growth invitro by hydroxyapatite nanoparticle‑delivered short hairpin RNAs against Stat3.

The present study investigated the effect of signal transducer and activator of transcription 3 (Stat3) interference on RM1 prostate cancer cell viability invitro, using plasmid‑based Stat3 specific short hairpin RNA (sh‑Stat3) delivered by hydroxyapatite nanoparticles (HAP). HAP carrying sh‑Stat3 plasmids were transfected into tumor cells. MTT assays were used to measure RM1 cell viability 24 and 48h following transfection, and the apoptosis rate and cell cycle phase distribution were determined by flow cytometry. Stat3 mRNA expression levels were measured by reverse transcription‑quantitative polymerase chain reaction and Stat3, CyclinD1, B cell lymphoma 2 apoptosis regulator (Bcl‑2), vascular endothelial growth factor (VEGF), Bcl‑2 associated Xapoptosis regulator (Bax) and cleaved‑caspase‑3 protein expression levels were detected using western blot analysis. The results demonstrated that HAP‑delivered sh‑Stat3 significantly decreased RM1 cell viability through the promotion of cell cycle arrest and apoptosis. Stat3 mRNA and protein expression levels were significantly downregulated in RM1 cells. Bcl‑2, VEGF and Cyclin D1 were also significantly downregulated, but cleaved‑caspase‑3 and Bax mRNA and protein expression levels were significantly upregulated. HAP‑delivered sh‑Stat3 decreased RM1 cell viability invitro, and HAP assisted plasmid‑based delivery of shRNA into tumor cells. The present results suggest that HAP may be a useful method for successful shRNA delivery into tumors.

Stat3 contributes to cancer progression by regulating Jab1/Csn5 expression.

Our previous studies demonstrated that Jab1/Csn5 overexpression is correlated with low survival rates in cancer patients including nasopharyngeal carcinoma (NPC), breast cancer, hepatocellular carcinoma, and contributes to NPC’s resistance to radiotherapy and cisplatin by regulating DNA damage and repair pathways. However, the molecular mechanism by which Jab1/Csn5 expression is upregulated in NPCs has yet to be determined. In the present study, we identified the upstream regulator of Jab1/Csn5 expression and demonstrated its role in intrinsic resistance of NPC cells to treatment with cisplatin. Signal transducer and activator of transcription-3 (Stat3) expression correlates with and contributes to Jab1/Csn5 transcription. Consistently, silencing of Stat3 in tumors reduced Jab1/Csn5 expression, thereby sensitizing NPC cells to cisplatin-induced apoptosis both in vitro and in vivo. Mechanistically, Stat3 transcriptionally regulated Jab1/Csn5. Further, high mRNA expression levels of Stat3 or Jab1 in colon cancer, breast cancer, and glioblastoma are associated with significantly shorter survival times from the R2 online database. These findings identify a novel Stat3-Jab1/Csn5 signaling axis in cancer pathogenesis with therapeutic and prognostic relevance.

Therapeutic Effect of Attenuated Salmonella Carrying SiRNA-STAT3 Plasmid on Transplanted Prostate Cancer in Mice

Objective To observe the therapeutic effect of attenuated Salmonella carrying siRNA-STAT3 plasmid on transplanted prostate cancer in mice, and investigate the mechanism of siRNA-STAT3 inducing apoptosis of prostate cancer cells in mice. Methods The transplanted prostate cancer models of mice were built, and then the mice were randomly divided into Mock group, pGC-Si-Scramble group and pGC-Si-STAT3 group. The general status of the mice was observed and the changes of tumor volume were recorded. The expression levels of mRNA and protein of STAT3 and its downstream genes such as Bcl-2, c-Myc, HIF-1, cyclinD1 were analyzed with RT-PCR and western blotting, and the apoptosis of tumor cells was detected with flow cytometry. Results Compared with the Mock group, the tumor weight and tumor volume in pGC-Si-STAT3 group were significantly decreased (P&lt;0.05). The result of flow cytometry revealed that the early apoptotic rate of tumor cells in pGC-Si-STAT3 group was (29.1±1.6)%, which was significantly higher than that of pGC-Si-Scramble group (14.7±1.4)% and Mock group (8.9±1.8)% (P&lt;0.05). The mRNA expression levels of STAT3 and its downstream genes Bcl-2 and c-Myc in the pGC-Si-STAT3 group were significantly decreased (P&lt;0.05), and the protein expression levels of STAT3 and its downstream genes HIF-1 and cyclinD1 were also decreased (P&lt;0.05), compared with the Mock group. Conclusion The attenuated Salmonella carrying siRNA-STAT3 plasmid could inhibit the growth of transplanted prostate cancer in mice by regulating the expressions of STAT3’s downstream genes and inducing the apoptosis of tumor cells.

Acitretin induces apoptosis and changes of relative signaling pathway in epidermoid carcinoma cell line A431

To study the effects of Acitretin on growth inhibition and apoptosis of epidermoid carcinoma cell line A431 and its molecular mechanisms. A431 cells were treated with Acitretin at the concentration of 10(-5)mol/L in different time intervals. The inhibition of cell growth was determined by MTT method, morphological changes were observed by electron microscopy, apoptosis was assessed by flow cytometry and Annexin-V staining. The mRNA expression levels of STAT3, cyclinD1 and p42/44MAPK were detected by reverse transcriptase polymerase chain reaction (RT-PCR). The protein expression levels of P-STAT3 and CyclinD1 were observed by Western blot in A431 cells. (1)Acitretin inhibited the growth of A431 cells in vitro in a dose-and time-dependent manner. Morphological changes revealed characteristics of cell apoptosis. Flow cytometry showed more sub-G(1) phase in A431 cells and more cells positively stained with Annexin-V. (2)Acitretin significantly inhibited the expression of STAT3 and CyclinD1 mRNA in A431 cells in vitro in a time-dependent manner(P<0.05). The p-STAT3 and CyclinD1 protein levels were down regulated. The Acitretin could also down regulate the p42/4MAPK mRNA in A431 cells. (3) After incubation with Acitretin, the mRNA level of CyclinD1 in A431 cells was positively correlated with that of STAT3(p<0.05). The protein level of CyclinD1 was also positively correlated with that of p-STAT3(p<0.05). However, there was no correlation between Mrna levels of CyclinD1 and p42/44MAPK. (1)Acitretin plays an inhibitory role in the tumor cell growth and induces the cell apoptosis in A431 cells. (2)The regulation of the Jak/STAT3 signaling pathway may play an important role in inducing growth inhibition and apoptosis by Acitretin in A431 cells.

Correlation between Stat3 signal transduction pathway and expression of cyclooxygenase-2 in colorectal cancer cells

To explore the expression of Stat3 signal transduction pathway and expression of cyclooxygenase-2 (COX-2) in colorectal cancer cells and their correlation with the clinicopathological parameters of colorectal cancer, and to reveal the mechanism of Stat3 signal transduction pathway regulating the expression of cCOX-2 in colorectal cancer cells. RT-PCR was used to detect the mRNA expression of Stat3 and COX-2 and immunohistochemistry was used to detect the protein expression of Stat3 and COX-2 in 50 specimens excised during operation from 50 patients with colorectal cancer aged 58 (37-75). Human colorectal cancer cells of the HT-29 strain were cultured. MTT method was used to examine the growth of the cells. AG490, an inhibitor of the upstream kinase JAK2 of Stat3 was added in to the culture fluid. 24, 48, and 72 hours later flow cytometry was used to examine the apoptosis of the cells. The mRNA expression levels of Stat3 and COX-2 in the colorectal cancer tissues were 1.97 and 1.88 times those of the adjacent normal tissues (both P < 0.05). The mRNA expression levels of Stat3 and COX-2 in the colorectal cancer tissues of the patients with lymph node metastasis were both significantly higher than those of the patients without lymph node metastasis (both P < 0.05). The mRNA expression levels of Stat3 and COX-2 in the colorectal cancer tissues of the patients of cancer with low differentiation were both significantly higher than those of the patients with high differentiation (both P < 0.05). Pearson correlation analysis showed that Stat3 mRNA expression was linearly correlated with COX-2 mRNA expression (r = 0.749, P < 0.01). The protein expression levels of Stat3 and COX-2 in the colorectal caner tissues were significantly higher than those in the adjacent normal tissues (both P < 0.01). AG490 time and dose-dependently inhibited the growth of the HKC cells, induced apoptosis of the HKC cells, and down-regulated the protein expression of Stas3 and COX-2. Overexpression of Stat3 and COX-2 may play a critical role in the development of colorectal cancer. Stat3 pathway influences the proliferation and apoptosis of colorectal cells and COX-2 gene expression. Blockade of the Stat3 signal pathway inhibits the proliferation and promotes the apoptosis of colorectal cancer cells.