JAK2 mRNA Research Articles

P3.01-039 JAK2 Participates in Lung Cancer Cells Proliferation, Migration and Invasion: Topic: Functional Biology in Lung Cancer

Lung cancer ranks as the first most common cancer and the first leading cause of cancer-related death in China and worldwide. Due to the difficulty in early diagnosis and the onset of cancer metastasis, the 5-year survival rate of lung cancer remains extremely low. JAK2 has emerged as pivotal participant in biological processes, often dysregulated in a range of cancers, including lung cancer. Recently we found that JAK2 might play an important role in lung cancer pathogenesis as an oncogene. While our understanding of JAK2 in the onset and progression of lung cancer is still in its infancy, there is no doubt that understanding the activities of JAK2 will certainly secure strong biomarkers and improve treatment options for lung cancer patients. The expression levels of JAK2 mRNA and protein were assayed using the RT-PCR and Western Blot assay respectively. MTT assay, Scratch-wound healing assay, Transwell migration and invasion assay were conducted to study the proliferation, migration and invasion abilities of lung cancer cells independently. The shRNA and overexpression plasmids of JAK2 were conducted. JAK2 is up-regulated in lung cancer tissues when compared with their adjacent non-tumor tissues, and was associated with lymph node metastasis. Downregulation of JAK2 inhibits the proliferation, migration and invasion abilities of lung cancer cells. Moreover, overexpression of JAK2 induced the proliferation, migration and invasion abilities of lung cancer cells. These findings demonstrate that JAK2, whose expression is up-regulated in lung cancer, may participate in lung cancer progression by regulating cancer cells proliferation, migration and invasion.

Injectable hyaluronic acid down-regulates interferon signaling molecules, IGFBP3 and IFIT3 in the bovine intervertebral disc.

The pro-inflammatory environment of the degenerated IVD represents a challenge for regenerative therapies. The study demonstrates that hyaluronan acts as an anti-inflammatory molecule by down-regulating IFNAR1 and IFNAR2, the signaling molecules STAT1, STAT2, JAK1 and the downstream apoptotic targets IGFBP3 and IFIT3. We also demonstrated that hyaluronan modulates the disc matrix environment by increasing aggrecan and collagen I synthesis and down-regulating ADAMTS4 that degrades the matrix under inflammatory conditions. The significance of this work lies in the fact that hyaluronan acts as an anti-inflammatory molecule by shifting the disc environment towards a more anabolic state and by promoting native IVD matrix production.

High mobility group box 1 induces the activation of the Janus kinase 2 and signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in pancreatic acinar cells in rats, while AG490 and rapamycin inhibit their activation.

The pathogenesis of severe acute pancreatitis (SAP) remains unclear. The Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway is important for various cytokines and growth factors. This study investigated the effect of the late inflammatory factor high mobility group box 1 (HMGB1) on the activation of JAK2/STAT3 in pancreatic acinar cells and the inhibitory effects of AG490 (a JAK2 inhibitor) and rapamycin (a STAT3 inhibitor) on this pathway. Rat pancreatic acinar cells were randomly divided into the control, HMGB1, AG490, and rapamycin groups. The mRNA levels of JAK2 and STAT3 at 10, 30, 60, and 120 minutes were detected using reverse transcription polymerase chain reaction (RT-PCR). The protein levels of JAK2 and STAT3 at 60 and 120 minutes were observed using Western blotting. Compared with the control group, the HMGB1 group exhibited significantly increased levels of JAK2 mRNA at each time point; STAT3 mRNA at 30, 60, and 120 minutes; and JAK2 and STAT3 proteins at 60 and 120 minutes (p < 0.01). Compared with the HMGB1 group, the AG490 and rapamycin groups both exhibited significantly decreased levels of JAK2 mRNA at each time point (p < 0.05); STAT3 mRNA at 30, 60, and 120 minutes (p < 0.01); and JAK2 and STAT3 proteins at 60 and 120 minutes (p < 0.01). HMGB1 induces the activation of the JAK2/STAT3 signaling pathway in rat pancreatic acinar cells, and this activation can be inhibited by AG490 and rapamycin. The results of this study may provide new insights for the treatment of SAP.

MiR-101 sensitizes K562 cell line to imatinib through Jak2 downregulation and inhibition of NF-κB target genes.

Imatinib mesylate (IM) is a frontline treatment in the early chronic phase of chronic myeloid leukemia (CML). However, intrinsic and acquired resistance against this drug has been defined and this issue has become a problem and a challenge in CML treatment. According to new findings, the inhibition of Janus kinase 2 (Jak2) in Bcr-Abl+ cells can promote apoptosis in IM-resistant cells. microRNAs (miRNAs) regulate the gene expression by targeting the messenger RNA (mRNA) for degradation. Recently, a growing body of evidence has implicated that dysregulation of miRNAs is associated with cancer initiation and development. In this report, we proposed that miRNA-101 targets Jak2 mRNA and regulates its expression and induces K562 leukemia cell apoptosis. Here, we transduced the K562 cell line with a miR-101-overexpressing vector and evaluated the Jak2 mRNA level. Our results showed that miR-101 overexpression in Bcr-Abl+ cells reduced the Jak2 mRNA level. Moreover, imatinib treatment and miR-101 upregulation led to miR-23a overexpression, which has putative binding site(s) on 3'-untranslated regions (3'-UTRs) of STAT5, CCND1, and Bcl-2 genes. Our results also indicated that miR-101 overexpression inhibited cell proliferation indicated by the MTT assay and promoted apoptosis detected via flow cytometry. Importantly, mRNA expression of NF-kappa B-regulated anti-apoptotic (Bcl-2, Bcl-xl, MCL-1, XIAP, and survivin) and proliferative (c-Myc and CCND1) genes was decreased. These findings suggest that miR-101 acts as a tumor suppressor by downregulating Jak2 expression and sensitizing K562 cells to imatinib. Therefore, restoration of miR-101 may be a therapeutic approach for CML treatment.

Synthetic Lethal Screen Demonstrates That a JAK2 Inhibitor Suppresses a BCL6-dependent IL10RA/JAK2/STAT3 Pathway in High Grade B-cell Lymphoma

We demonstrate the usefulness of synthetic lethal screening of a conditionally BCL6-deficient Burkitt lymphoma cell line, DG75-AB7, with a library of small molecules to determine survival pathways suppressed by BCL6 and suggest mechanism-based treatments for lymphoma. Lestaurtinib, a JAK2 inhibitor and one of the hits from the screen, repressed survival of BCL6-deficient cells in vitro and reduced growth and proliferation of xenografts in vivo BCL6 deficiency in DG75-AB7 induced JAK2 mRNA and protein expression and STAT3 phosphorylation. Surface IL10RA was elevated by BCL6 deficiency, and blockade of IL10RA repressed STAT3 phosphorylation. Therefore, we define an IL10RA/JAK2/STAT3 pathway each component of which is repressed by BCL6. We also show for the first time that JAK2 is a direct BCL6 target gene; BCL6 bound to the JAK2 promoter in vitro and was enriched by ChIP-seq. The place of JAK2 inhibitors in the treatment of diffuse large B-cell lymphoma has not been defined; we suggest that JAK2 inhibitors might be most effective in poor prognosis ABC-DLBCL, which shows higher levels of IL10RA, JAK2, and STAT3 but lower levels of BCL6 than GC-DLBCL and might be usefully combined with novel approaches such as inhibition of IL10RA.

Investigaion about the mRNA expression of ASB2 and Jak3 in patients with acute lymphoblastic leukemia

Objective To explore the expression of ankyrin-repeat SOCS box containing protein 2 (ASB2) and Janus kinase 3 (Jak3) mRNA in bone marrow plasma mononuclear cells of patients with acute lymphoblastic leukemia (ALL) and discuss the correlation of them. Methods Bone marrow plasma was collected from 48 patients of newly diagnosed ALL (37 cases B-ALL and 11 cases T-ALL) and 34 non leukemia patients (control group), respectively. Expression levels of ASB2 and Jak3 mRNA were detected by real-time quantitative PCR. Results The expression of ASB2 mRNA in B-ALL and T-ALL were significantly different compared to control group (t=14.2, P<0.01; t=15.2, P<0.01), which were 68.5, 32.7 folded more than control group, respectively. Moreover, the expression level of Jak3 mRNA in B-ALL group and T-ALL group were 2 336.1 and 7 131.5 folded more than control group (t=37.3, P<0.01; t=37.6, P<0.01). At last, the expression of ASB2 and Jak3 gene was positive correlation (r=0.523, P<0.001). Conclusion ASB2 and Jak3 are expressed abnormally in ALL patients and the correlation of them is positive. ASB2 and Jak3 may be related to the proliferation and differentiation in leukemia cells. Key words: Leukemia, lymphoid; Bone marrow; Polymerase chain reaction; Ankyrin-repeat SOCS box containing protein 2; Janus kinase 3

Acute Monocytic Leukemia Associated Antigen MLAA-34 up-Regulates JAK2/STAT3 Expression and JAK2/STAT3 Enhances MLAA-34 Activation in a Positive Feedback Loop

Backgroud: The MLAA-34 gene (GenBank no. AY288977.2) was first discovered in acute monocytic leukemia (M5) in an effort to identify monocytic leukemia-associated antigens by serologic analysis of a recombinant cDNA expression library (SEREX). Previous study showed that high MLAA-34 levels were independently associated with a poorer relapse-free survival and overall survival in AML patients. The MLAA-34 is located on 13q14.2 and has been confirmed to be a novel splice variant of CAB39L (calcium binding protein 39-like). Both mRNA and protein levels of MLAA-34 were found to be higher in U937 cells and M5 patients. The study confirmed that MLAA-34 plays a role in the antiapoptosis of U937 cells, and has the function of oncogenes.Objective: This study is to explore the relationship of MLAA-34 and JAK2/STAT3 signaling pathway so as to further clarify antiapoptotic mechanisms of MLAA-34.Method: Potential binding sites for STAT3 was identified by computer-assisted analysis of the core promoter of MLAA-34 gene. We analyzed the role of STAT3 in the regulation of MLAA-34 gene expression in U937 cells by site-specific mutation, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). The expression of MLAA-34 was detected by RT-PCR and western blot after over-expressing and interfering STAT3. Through the different concentrations of AG490 (JAK2 inhibitors) and different concentrations of IL- 6 (JAK2 activator) study JAK2/STAT3 signaling pathway upregulated MLAA-34 expression. The gene chip and co-IP were applied to study the MLAA-34-induced JAK2/STAT3 activation. Peripheral blood mononuclear cells and bone marrow (BM) cells of M5 patients were obtained. In M5 patients, mRNA and protein expression of JAK-2, STAT3, p-STAT3 and MLAA-34 were determined by quantitative RT-PCR and western blot respectively to verify the forward feedback regulation pathway.Result: The reporter assay showed that the activity of reporter gene was downregulated after the mutation of STAT3 binding sites. ChIP assay and EMSA showed that STAT3 can directly bind to MLAA-34 gene promoter. The expression vectors of MLAA-34 as well as siRNA eukaryotic expression vectors respectively targeting STAT3 were successfully constructed. RT-PCR and western blot results showed that STAT3 can increase the level of MLAA-34. Research of administration of different concentrations of AG490 and different concentrations of IL-6 showed that JAK2/STAT3 signaling pathway could upregulate MLAA-34 expression. AML-M5 NOD / SCID mice leukemia model was successfully constructed,.Konckdown of MLAA-34 gene can promote apoptosis of leukemia cells, inhibit tumor growth and prolong survival time. Total RNA from shRNA-MLAA-34/U937 and Vec/U937 cells were analyzed by Human Genome U133 chip. Heat-map showed reduced expression of JAK2/STAT3 pathway genes. The result was verified by qPCR and western blot. CO-IP showed that MLAA-34 could form a complex with endogenous JAK2 under the enhanced role of IL-6. Thereby activating JAK2 may directly or indirectly dependent on the presence of MLAA-34. Furthermore, we detected JAK-2, STAT3, P-STAT3 and MLAA-34 gene and protein level in M5 patients, the results showed that they have a positive correlation.Conclusion: JAK2/STAT3 pathway up-regulates MLAA-34 transcription, and MLAA-34 enhances JAK2/STAT3 pathway activation. The forward feedback regulation may have profound therapeutic implications for M5 and could help invent novel approaches in treatment for acute monocytic leukemia. DisclosuresNo relevant conflicts of interest to declare.

Abstract 12229: MicroRNA Regulation of JAK-STAT System in the Atrial Fibrillation-Related Fibrotic Response

Introduction: MicroRNAs (miRNAs) are involved in cardiac remodeling, but their role in atrial fibrillation (AF) is poorly understood. JAK-STAT signalling is activated by the pro-fibrotic mediator PDGF, but its contribution to AF substrate formation is unknown. Here, we investigated miRNA regulation of the JAK-STAT system in in the heart failure (HF) induced AF fibrotic substrate. Methods: HF was induced in dogs by ventricular tachypacing (VTP, 240 bpm) for 2 weeks. Fibroblasts (FBs) isolated from the left atrium (LA) and left ventricle (LV) of control (CTL) dogs were treated with PDGF-AB. PDGF, JAK2, STAT3 and miRNA expression was assessed by qPCR. JAK2, STAT3 and collagen-I (COL-1) proteins were quantified by Western Blot. miRNA targets were validated by luciferase reporter assay. miRNA mimic or antisense oligonucleotides (AMOs) were used for miRNA manipulation in FBs. Results: HF dogs developed AF susceptibility and LA-selective fibrosis beginning at 1 wk VTP. PDGF and JAK2 mRNA, along with phosphorylated (activated) JAK2 protein, were increased in HF atria (by 1.4-2.9* fold, *p&lt;0.05). miR-30a and miR-133a were predicted bioinformatically to target JAK2, and were downregulated by 51%-52%** and 48%-71%** (**p&lt;0.01) in HF LA FBs at 1-2 wk VTP. PDGF-AB increased the expression of JAK2 and STAT3 mRNA (by 1.4-1.5* fold) and COL-1 protein (by 2*** fold, ***p&lt;0.001) in canine LA FBs, while reducing the expression of miR-30a and miR-133a (by ~50%-80% ***). Smaller changes were seen in LV FBs with PDGF-stimulation. Luciferase assay confirmed miR-30a and miR-133a targeting of JAK2. Overexpression of miR-133a downregulated JAK2 mRNA (by 30%***) and protein (by 50%, p=0.05) in LA FBs. This effect was abolished by co-transfection of miR-133a and AMO-133a. The upregulation of JAK2 and COL-1 expression induced by PDGF-AB stimulation was reversed by miR-133a overexpression in LA FBs (reduced by 49%* and 53%*** versus PDGF stimulation alone). Conclusions: PDGF-induced disinhibition of JAK2 expression by downregulation of miR-30a and miR-133a may contribute to AF-associated fibrotic responses. MicroRNA targeting of the JAK-STAT system regulates LA FB function, appears to be involved in HF-induced LA fibrosis and is a potential therapeutic target for AF prevention.

Abstract 12040: JAK-STAT Signalling and the Atrial Fibrillation-Promoting Fibrotic Substrate: Pathogenic Role and Possible Therapeutic Target

Introduction: Left atrial (LA) fibrosis is an important feature of the atrial fibrillation (AF) substrate. The JAK-STAT system, activated by platelet derived growth factor (PDGF), contributes to cardiac remodeling but its role in AF is unknown. Here, we investigated the participation of the JAK-STAT system in LA fibrosis development. Methods: Heart failure (HF) was induced in dogs by ventricular tachypacing (VTP, 240 bpm) and in mice subjected to myocardial infarction (MI). Protein and mRNA expression were measured in freshly isolated LA fibroblasts (FBs) by Western blot and qPCR. Mice received S3I-201,a selective STAT3 inhibitor, or vehicle control (CTL) by osmotic minipump for 2 weeks post MI. Results: HF dogs developed progressive LA-selective fibrosis and AF susceptibility beginning at 1 wk VTP. HF increased JAK2 mRNA expression in LA FBs from 1 wk VTP (1.4-1.5 fold**, **P&lt;0.01). Phosphorylated JAK2 and STAT3 protein increased in HF atrial tissue by 2.5** and 2.9* fold (*p&lt;0.05) respectively. Upstream signalling through PDGF was also enhanced by HF: PDGF A/C/D upregulated by 2.2-3.5*** fold (***p&lt;0.001); PDGFR β upregulated by 2.3** fold. PDGF stimulation of CTL dog LA FBs increased TGFβ1, TGFβ receptor 2, STAT3, collagen 1α1 and collagen 3α1 expression (by 1.2-2.3 fold***). These effects were abolished by S3I-201 in a dose dependent manner (by 79%*** at 100 μM). In vivo S3I-201 treatment (Figure) decreased LA fibrosis (by 60%***), LA dimension at end systole (by 11%*) and P wave duration (by 31%***). MI-induced LV dilation and contractile dysfunction were not affected by S3I-201. Conclusions: HF activates the LA JAK-STAT system via enhanced PDGF signalling. JAK-STAT inhibition reduces the profibrotic effects of PDGF stimulation on canine FBs in vitro and attenuates LA fibrosis and electrical remodeling post MI in mice. These results suggest that the JAK/STAT pathway plays an important role in LA fibrogenesis and is a potential target for LA fibrosis prevention in man.

Cover Photograph: Xenopus spermatogonial stem cells (SSCs) at telophase. Primary spermatogonium destined to differentiate and SSC are thought to arise from areas in which JAK1 mRNA (blue signals in the middle and right column) is present (arrowheads) and absent (double arrowheads), respectively. (See Hyakutake et al.)

Development, Growth & DifferentiationVolume 57, Issue 5 p. i-i Cover ImageFree Access Cover Photograph: Xenopus spermatogonial stem cells (SSCs) at telophase. Primary spermatogonium destined to differentiate and SSC are thought to arise from areas in which JAK1 mRNA (blue signals in the middle and right column) is present (arrowheads) and absent (double arrowheads), respectively. (See Hyakutake et al.) First published: 09 June 2015 https://doi.org/10.1111/dgd.12169AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume57, Issue5June 2015Pages i-i RelatedInformation

Asymmetrical allocation of JAK1 mRNA during spermatogonial stem cell division in Xenopus laevis.

During Xenopus spermatogenesis, each primary spermatogonium (PG), the largest single cell in the testis, undergoes mitotic divisions with a concomitant decrease in size to produce smaller differentiating spermatogonia. The spermatogonial stem cells (SSCs) occur in this PG population. Taking advantage of identifiable and isolatable properties of Xenopus SSCs, we examined JAK1 gene expression during the spermatogenesis because there have been reports on the important role of JAK/STAT pathway in regulating the status of SSCs in Drosophila and mouse. Surprisingly, insitu hybridization revealed the presence of JAK1 mRNA in the differentiating spermatogonia and primary spermatocytes as well as some PGs. Inhibition of JAK1 activity in the testis caused a decrease in percentage of BrdU-incorporating spermatogonia, suggesting that JAK1 was at least involved in regulation of spermatogonial proliferation. Interestingly, single cell reverse transcription-polymerase chain reaction (RT-PCR) clearly showed two different types of SSCs: SSCs with JAK1 mRNA (JAK1+ ) or without JAK1 mRNA (JAK1- ). Since JAK1- SSC level was increased by induction of testis regeneration, self-renewing SSCs were thought to be JAK1- . In addition, we found barrel-shaped PGs, in which JAK1 mRNA was localized asymmetrically to one half of the cell. The stainability with propidium iodide and morphology of two nuclei in the barrel-shaped PG were similar to those of PG nucleus. Based on the above observations, we propose the hypothesis that JAK1+ SSC is preparing for production of PGs destined to differentiate (destined PGs) and the accumulated JAK1 mRNA in the SSC is distributed exclusively into the destined PGs through mitotic division.

R-value comprehensive evaluation of effect of three methods for lung-kidney comprehensive evaluation study on R value of three methods for regulating and invigorating lung and kidney in regulating lung inflammation signaling pathways in COPD rats

Previous studies showed that three methods for regulating and invigorating lung and kidney (lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, and Qi supplementing and kidney nourishing) could regulate inflammatory signaling pathways of chronic obstructive pulmonary disease (COPD) in rats, so as to alleviate inflammation. In the present study, R-value comprehensive evaluation method was used to evaluate the comprehensive effect of three methods for regulating and invigorating lung and kidney on inflammatory signaling pathways. Rats were randomly divided into control, model, lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing and aminophylline groups. The COPD rat models were established by cigarette smoking combined with bacterial infection, and orally administered with drugs between the 9th and 20th week. Afterwards, efforts were made to observe the long-term effects between the drug withdrawal and the 32rd week and detect indicators in two batches in the 20th week and 32th week. Specifically, (1) Linking JAK/STAT signaling pathway: JAK2 mRNA, and protein expressions of STAT-1, STAT-3, STAT-5, JAK-2; (2) NF-kappaB signaling pathway: Smad2 mRNA and protein expressions of I-kappaB, NF-kappaB, TGF-beta1; (3) PPARgamma and antioxidant signaling pathway: SOD, PGE mRNA, PPARgamma protein. According to the results, 5 indicators in JAK/STAT pathway, 4 indicators in NF-kappaB pathway, and 3 indicators in PPARgamma pathway were significantly rectified by three methods for regulating and invigorating lung and kidney in between the 20th week and 32nd week. Between the 20th and 32nd week, the recipes for rectifying JAK/STAT pathway with intensity from high to low were recipes for lung invigorating and spleen strengthening, Qi supplementing and kidney nourishing, lung invigorating and kidney tonifying, aminophylline, particularly those for lung invigorating and spleen strengthening; The recipes for rectifying NF-kappaB pathway with intensity from high to low were recipes for lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing and aminophylline, particularly the first three types of drugs. The recipes for rectifying PPARgamma and antioxidant signaling pathway with intensity from high to low were recipes for lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing, lung invigorating and spleen strengthening and aminophylline. Therefore, three methods for regulating and invigorating lung and kidney showed better long-term effects in regulating COPD lung inflammation signaling pathways. Specifically, recipe for lung invigorating and spleen strengthening showed a better effect in JAK/STAT and NF-kappaB pathways, while recipe for lung invigorating and kidney tonifying and Qi supplementing and kidney nourishing showed better effects in PPARgamma and antioxidant signaling pathways. In conclusion, R-value comprehensive evaluation method can evaluate the comprehensive effect of medicines and define the ranking of multiple drugs and their main targets.

Effect of Liuweibuqi capsule, a Chinese patent medicine, on the JAK1/STAT3 pathway and MMP9/TIMP1 in a chronic obstructive pulmonary disease rat model

ObjectiveTo observe effect of Liuweibuqi Capsule, a Traditional Chinese Medicine (TCM), on the janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and matrix metalloproteinases (MMPs) in a chronic obstructive pulmonary disease (COPD) rat model with lung deficiency in terms of TCM's pattern differentiation. MethodsRats were randomly divided into a normal group, model group, Liuweibuqi group, Jinshuibao group, and spleen aminopeptidase group (n= 10). Aside from the normal group, all rats were exposed to smoke plus lipopolysaccharide tracheal instillation to establish the COPD model with lung deficiency. Models were established after 28 days and then the normal and model groups were given normal saline (0.09 g/kg), Liuweibuqi group was given Liuweibuqi capsule (0.35 g/kg), Jinshuibao group was given Jinshuibao capsules (0.495 g/kg), and the spleen group was given spleen aminopeptidase (0.33 mg/kg), once a day for 30 days. Changes in symptoms, signs, and lung histology were observed. Lung function was measured with a spirometer. Serum cytokines were detected using enzyme-linked immunosorbent assay, and changes in the JAK/STAT pathway, MMP-9, and MMPs inhibitor 1 (TIMP1) were detected by immunohistochemistry, RT-PCR, and western blotting, respectively. ResultsCompared with the normal group, lung tissue was damaged, and lung function was reduced in the model control group. Additionally, the levels of interleukin (IL)-1β, γ interferon (IFN-γ), and IL-6 were higher, while IL-4 and IL-10 were lower in the model control group than those in the normal group. The expressions of JAK1, STAT3, p-STAT3, and MMP-9 mRNA and protein in lung tissue were higher, and TIMP1 mRNA and protein was lower in the model group compared with the normal group. After treatment, compared with the model group, the expression of inflammatory cytokines was lower in each treatment group, and expressions of JAK/STAT pathway, MMPs were lower. Compared with the positive control groups, the Jinshuibao and spleen aminopeptidase groups, lung function was better, and JAK1, STAT3, and p-STAT3 protein were lower and TIMP1 was higher in the Liuweibuqi group. ConclusionLiuweibuqi capsules can improve the symptoms of COPD possibly by regulating the expression of the JAK1/STAT3 pathway and MMP9/TIMP1.

JAK2 Is a Direct BCL6 Target Gene: Implications for Therapy in Diffuse Large B-Cell Lymphoma

Increased STAT3 signalling is a factor in driving ~50% of diffuse large B-cell lymphoma. In some cases increased cytokine production by the lymphoma is responsible for activation of JAK2 and STAT3 but the regulation of signalling through this pathway is not clear. We constructed a conditional BCL6 deficient cell line through disruption of the endogenous BCL6 loci of a genetically tractable human B-cell lymphoma by homologous recombination, and insertion of a tetracycline regulatable BCL6 transgene. On induction of BCL6 deficiency growth of the cell line slowed by 3 to 4-fold. A synthetic lethal screen employing a library of small molecule inhibitors in genetically BCL6 deficient lymphoma cells showed that lestaurtinib, a JAK2 inhibitor, enhanced loss of viability. We investigated the hypothesis that JAK2 is a direct BCL6 target gene. JAK2 mRNA and protein expression were induced by BCL6 deficiency. Inspection of the JAK2 proximal promoter region demonstrated a potential BCL6 binding site. BCL6 bound to this sequence in vitro and mutagenesis of the binding site relieved BCL6 mediated transcriptional repression in luciferase reporter assays. Analysis of ChIP-seq data showed a binding peak in the JAK2 proximal promoter region confirming in vivo BCL6 binding. Data from a large and publicly available gene expression dataset confirmed an inverse correlation between BCL6 and JAK2 mRNA whilst there was a positive correlation between JAK2 and STAT3 mRNA. STAT3 is a known target of BCL6 transcriptional repression. We suggest that BCL6 represses both JAK2 and STAT3 and that relatively high BCL6 levels will tend to reduce JAK2-STAT3 signalling whereas lower BCL6 levels will amplify this pathway. Mouse xenografts utilising our conditional BCL6 deficient cell line showed that lestaurtinib alone caused minimum reduction to tumour size but the combination of BCL6 deficiency and JAK2 inhibitor caused growth reduction with central necrosis. In summary we identify JAK2 as a BCL6 target gene and propose that BCL6 has an important role in regulation of JAK2-STAT3 signalling in DLBCL. BCL6 mRNA expression may be a biomarker to enable the rational use of JAK2 inhibitors in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Abstract 776: Study on the induced resistance reversal by JAK2 gene RNAi and inhibitor AG490 in ovarian cancer paclitaxel-resistant cells

Abstract Objective: To explore the influence on OC3/TAX300 cell line and the change of chemosensitivity to paclitaxel after inhibiting JAK2 gene expression by constructing a recombined lentivirus vector which expressing small interfering RNA(siRNA) of JAK2 gene, and using JAK2 inhibitor AG490 to discuss the possible mechanism of JAK2 on ovarian cancer chemotherapy resistance Methods: Design and build JAK2 siRNA lentivirus vector by using RNA interference technology, and to transfect the drug resistant cell line OC3/TAX300. The expression levels of JAK2 was detected by RT-PCR and Western blotting. The cell proliferation condition was determined by MTT method, the cell ultrastructure change was observed by transmission electron microscope and the cell cycle and apoptosis were detected by flow cytometry; Western blot was used to detect the expression of STAT3 protein which is JAK2 downstream signal transduction factor. Use different concentration of AG490 solution to culture paclitaxel-resistant cell line OC3/TAX300 of ovarian cancer; JAK2 and STAT3 mRNA and protein expression level were detected by Real-time PCR and Western blot. Detect cell proliferation condition, observe the cell ultrastructure change and detect the cell cycle and apoptosis after treating the variety concentration of AG490 cultivate cells by taxol. Results: 1.The JAK2 siRNA could significantly inhibits the expression of JAK2 mRNA and protein (P&amp;lt;0.05) which confirmed by real-time PCR and western blot results. 2. MTT results showed that the cell growth of JAK2 gene silencing group was significantly inhibited by taxol treatment after 48h and 72h (all P&amp;lt;0.01); AG490 could enhance the sensitivity of cells to taxol(P&amp;lt;0.05); 3. Typical apoptotic cells could be found under the transmission electron microscope in both JAK2 gene silencing group and AG490 culture group; 4. Flow cytometry detection results showed that both the cell apoptosis rate and the cell cycle in G2/M phase proportion increased significantly in JAK2 gene silencing group (P&amp;lt;0.01); The application AG490 can make obviously increase of the cell apoptosis and the cell cycle in G2/M phase proportion induced by paclitaxel(P&amp;lt;0.05).5. Western blot results showed the expression level of STAT3 protein decreased after JAK2 gene silencing(P &amp;lt; 0.05). The expression of JAK2 and STAT3 mRNA and protein were inhibited with varying degrees after using different concentration of AG490 to culture cells(P&amp;lt;0.05).Conclusion: RNAi technology and using tyrosine kinase inhibitor AG490 can effectively restrain the JAK2 and STAT3 expression in ovarian cancer resistant cells. By restoring the paclitaxel effect on cell cycle block, AG490 could inhibit the cell proliferation, induce the cell apoptosis and strengthen the resistant cells of paclitaxel chemotherapy sensitivity. JAK2-STAT3 signal transduction pathway may be involved in the process. Note: This abstract was not presented at the meeting. Citation Format: Hongxia Li. Study on the induced resistance reversal by JAK2 gene RNAi and inhibitor AG490 in ovarian cancer paclitaxel-resistant cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 776. doi:10.1158/1538-7445.AM2014-776

IL-17A Alone Weakly Affects the Transcriptome of Intestinal Epithelial Cells but Strongly Modulates the TNF-α–induced Expression of Inflammatory Mediators and Inflammatory Bowel Disease Susceptibility Genes

In contrast to anti-TNF-α antibodies, anti-IL-17A antibodies lacked clinical efficacy in a trial with patients suffering from Crohn's disease. We therefore analyzed how IL-17A modulates the inflammatory response elicited by TNF-α in intestinal epithelial cells (IEC). Target mRNA levels in IEC and colonic biopsies were assessed by RNA microarray and quantitative real-time PCR. Signaling pathways were analyzed using receptor neutralization and pharmacological inhibitors. Target protein levels were determined by immunoblotting. Microarray analysis demonstrated that IL-17A alone is a weak inducer of gene expression in IEC (29 regulated transcripts), but significantly affected the TNF-α-induced expression of 547 genes, with strong amplification of proinflammatory chemokines and cytokines (>200-fold increase of CCL20, CXCL1, and CXCL8). Interestingly, IL-17A differentially modulated the TNF-α-induced expression of several inflammatory bowel disease susceptibility genes in IEC (increase of JAK2 mRNA, decrease of FUT2, ICAM1, and LTB mRNA). Negative regulation of ICAM-1 by IL-17A was verified on protein level. The significance of these findings is emphasized by inflamed lesions of patients with inflammatory bowel disease demonstrating significant correlations (P < 0.01, Rho, 0.57-0.85) for JAK2, ICAM1, and LTB mRNA with IL17A and TNF mRNA. Our study demonstrates the modulation of inflammatory bowel disease susceptibility gene mRNA in IEC as a novel important property of IL-17A. Given the weak impact of sole IL-17A stimulation on IEC target gene expression, our study provides an important explanation for the lack of clinical efficacy of sole IL-17A neutralization, but suggests a beneficial effect of combined IL-17A/TNF-α that is currently in clinical development.

STAT3 inhibitor WP1066 as a novel therapeutic agent for bCCI neuropathic pain rats

Activation of signal transducer and activator of transcription-3 (STAT3) is suggested to be critically involved in the development of chronic pain, but the complex regulation of STAT3-dependent pathway and the functional significance of inhibiting this pathway during the development of neuropathic pain remain elusive. To evaluate the contribution of the JAK2/STAT3 pathway to neuropathic pain and the potentiality of this pathway as a novel therapeutic target, we examined the effects of the STAT3 inhibitor WP1066 by intrathecal administration in a rat model of bilateral chronic constriction injury (bCCI). The pain behavior tests were performed before the surgery and on postoperative day 3, 7, 14 and 21. L4–L6 dorsal spinal cord were harvested at each time point. Both RT-PCR and Western blot were performed to evaluate the activation of JAK2/STAT3 pathway. To observe the influence of WP1066 on neuropathic pain and its molecular mechanism, WP1066 (10μl, 10mmol/L in DMSO) or the same capacity of DMSO as the control were applied through the intrathecal tube on the day before bCCI surgery, and on the postoperative day 3 and 5. Behavioral tests were performed to observe the therapeutic effect on mechanical, thermal and cold hyperalgesia. L4–L6 dorsal spinal cord was harvested on postoperative day fourteen, followed by RT-PCR and Western blot evaluation of the JAK2/STAT3 pathway activation.The mechanical, thermal and cold hyperalgesia of the bCCI rats were significantly decreased when compared with the Sham or the Naïve group at each postoperative time point (P<0.05). JAK2 mRNA and STAT3 mRNA were significantly increased in the bCCI rats, accompanied by SOCS3 mRNA with a similar tendency. Western blot analysis showed that JAK2 and phosphorylated STAT3 increased significantly since 3 days after bCCI. JAK2 peaked on postoperative day 14 while phosphorylated STAT3 peaked on postoperative day 7 and gradually decreased thereafter and SOCS3׳s peak level on postoperative day 3. When WP1066 were administered intrathecally, the pain behaviors of the bCCI rats were significantly improved (P<0.05). WP1066 also inhibited the mRNA level of JAK2, STAT3 and SOCS3 in bCCI rats significantly, together with the protein level of JAK2, phosphorylated STAT3 and SOCS3 on postoperative day 14 as well.Our results found that the JAK2/STAT3 pathway in the spinal cord dorsal horn was significantly activated in the bCCI neuropathic pain rats. WP1066, which inhibited the STAT3 pathway specifically, could partially alleviate the pain behavior of the bCCI rats. So it may serve as a novel therapeutic strategy against neuropathic pain.

Bortezomib induces apoptosis by interacting with JAK/STAT pathway in K562 leukemic cells.

In the current study, we aimed to identify the cytotoxic and apoptotic effects of bortezomib (BOR) on human K562 chronic myelogenous leukemia cells and to evaluate the potential roles of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway members STAT3, STAT5, and JAK2 on BOR-induced cell death of leukemic cells. Cell viability was assessed via trypan blue dye exclusion test, and cytotoxicity of the BOR-treated cells was conducted by 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay. The relative messenger RNA (mRNA) expression levels of STAT3, STAT5A, STAT5B, and JAK2 were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). On the other hand, their protein expression levels were detected by western blot method. The obtained results indicated that BOR treatment reduced cell viability and induced leukemic cell apoptosis in a dose- and time-dependent manner as compared to untreated control cells. While mRNA expression levels of STAT5A, STAT5B, and STAT3 were significantly reduced following BOR treatment when compared to untreated controls, it had no effect upon JAK2 mRNA expression. As for protein levels, STAT expressions were downregulated after BOR treatment especially at 72nd and 96th hours. Our results pointed out that BOR treatment had a significant potential of being an anticancer agent for chronic myelogenous leukemia therapy, and this effect could be due to the expressional downregulations of JAK/STAT pathway members.

Alterations in Janus (JAK2)/signal transducer of activated transcription signaling pathway in human renal cell carcinoma (1179.2)

RCC accounts for 9 out of 10 cases of kidney cancer. Approximately 65,150 new cases and 13,680 deaths are expected from this disease in 2013. There is urgent clinical need for better treatment options as RCC responds poorly to current chemotherapy. A major limitation to developing new treatments is that the molecular mechanisms responsible for inappropriate cell survival and chemoresistance are unknown. One pathway suggested to be involved in vitro is the JAK2/STAT. We hypothesized that there would be altered expression of the members and regulators of the JAK/STAT pathways in RCC. We utilized samples from normal (n=14), benign (n=6), and histological Fuhrman grades: Grade 1 (n=3), Grade 2 (n=10), Grade 3 (n=14), and Grade 4 (n=3). We analyzed 31 human RCC samples as well as the normal and benign samples with Affymetrix human gene microarrays and Western Blot technology. The samples were all clear cell renal carcinomas verified by a Beaumont Hospital pathologist. We analyzed 22,148 genes and altered gene expression was then detected by ANOVA taking into account histological stage. We found 170 genes that were differentially expressed in all grades compared to both normal and benign kidney samples. We found both JAK1 and JAK2 mRNA to be highly expressed in the RCC samples compared to the controls. We did not observe any decrease in the mRNA levels or protein expression of the regulatory proteins SOCS1, SOCS3 or SHP‐1. We did find an increase in SHP‐1 serine phosphorylation which is inhibitory in the RCC samples. These data suggest that altered function of the regulators not expression may be involved in RCC.Grant Funding Source: Zafarna Foundation

Effect of arsenic trioxide and 5-aza-2'-deoxycytidine on SHP-1, JAK3, TYK2 gene expression in K562 cells

This study was purposed to explore the effects of a methylation inhibitor arsenic trioxide (As2O3, ATO) and 5-Aza-2'-deoxycytidine (5-aza-CdR) on the expression of JAK-STAT signal transduction pathway in family members JAK3, TYK2 and hematopoietic cell phosphatase SHP-1 in chronic myeloid leukemia cell line K562 and their roles in pathogenesis of leukemia. The K562 cells were divided into 3 groups:single drug-treated group, combined 2 drugs-treated group, group without drug treatment as control. The concentration of 5-aza-CdR were 0.5, 1, 2 µmol/L; the concentration of ATO was 1, 2.5, 5 µmol/L; the concentration of combined drugs was ATO 1 µmol/L + 5-aza-CdR 0.5 µmol/L, ATO 2.5 µmol/L + 5-aza-CdR 1 µmol/L, and ATO 5 µmol/L + 5-aza-CdR 2 µmol/L. The K562 cells were treated with above-mentioned concentration of drugs for 24, 48 and 72 hours, then the total RNA of cells was extracted, the JAK3, TYK2 and SHP-1 expressions were detected by real-time quantitative-PCR. The results showed that after the K562 cells were treated with ATO and 5-aza-CdR alone and their combination, the expression of SHP-1 mRNA increased, the expressions of JAK3 mRNA and TYK2 mRNA decreased along with increasing of concentration and prolonging of time, displaying the concentration and time-dependency. The SHP-1 negatively related with JAK3 and TYK2. The effect of SHP-1 on JAK3 was significantly higher than that on TYK2. It is concluded that when the K562 cells are treated with ATO and 5-aza-CdR alone and their combination, the expression of SHP-1 is up-regulated and the expressions of JAK3, TYK2 are down-regulated in concentration-and time-dependent manners, moreover the ATO and 5-aza-CdR show synergies demethylation effect. The SHP-1 gene exert effect possibly through inhibiting the JAK/STAT pathway, the JAK3 is affected more than TYK2, the JAK3 may exert more important role in TAK/STAT pathway.