Results Of Electrophoretic Mobility Shift Assay Research Articles

Molecular Genetic Study on HAND2 Gene Promoter in Ventricular Septal Defect.

Ventricular septal defect (VSD), the most common type of congenital heart disease (CHD), is primarily caused by cardiac dysplasia. Heart and neural crest derivatives expressed 2 (HAND2) participates in developing the right heart. The loss of HAND2 expression in humans is closely connected with ventricular septal defects. We used a case-control study to analyze the genetic variations in the HAND2 promoter region in VSD patients and controls. Some statistical analysis methods were used to analyze the association of single nucleotide polymorphisms (SNPs) with VSD. The dual-luciferase reporter assay and electrophoretic mobility shift assay (EMSA) were used to conduct functional analysis and molecular mechanism study of genetic variations. Through sequencing, we identified nine genetic variants in patients with VSD. The SNP rs2276940 G>T and rs2276941 G>A were associated with an increased risk of VSD. The dual-luciferase reporter assay showed that SNP rs2276940 G>T and rs138531627 C>G decreased the transcriptional activity of the HAND2 promoter. Transcription factors (TFs) predicting suggested that all three SNPs may change the binding of TFs. The result of EMSA showed that rs138531627 C>G may create a new binding site for TFs while rs2276940 G>T enhanced the binding affinity for TFs. These results indicated that genetic variants of the HAND2 promoter may increase the risk of VSD, and the molecular mechanism may be the change of the binding affinity of TFs.

Pregnancy Hormone Mediated Tumorigenesis in BRCA1 Defective Breast Cancers

Background and ObjectiveBRCA1 mutations predispose to breast and ovarian cancers, the exact reason for the tissue specificity is unknown. The role of hormonal factors estrogen/estrogen receptor α (ER‐α) was hypothesized to be a major contributing factor for this tissue specificity. However, tumor progression in BRCA1 defective condition could not be controlled by inhibiting ER‐α as they either lack or exhibit very low expression of ER‐α, progesterone receptor, and human epidermal growth factor receptor. In addition to these hormone receptors, the prominent hormone known to be important for the development and differentiation of breast tissue is the Human Chorionic Gonadotropin (hCG). Thus, we speculated the role of hCG, especially the β‐hCG, to be the key molecule actively contributing to the tumorigenesis in TNBCs.In our previous study, we have identified for the first time that β‐hCG expression is linked to BRCA1 status. β‐hCG overexpression was observed in BRCA1 mutated breast cancer cells and conditional knockout mouse models. ChIP and luciferase assay also demonstrated the transcriptional regulation of BRCA1 over β‐hCG. β‐hCG mediate signaling through TGFβRII and promotes migration and invasion predominantly in BRCA1 mutated breast cancer cells (Sengodan et al., 2017). In accordance with the above findings, the present study aims to analyze the effectiveness of β‐hCG as a potential diagnostic marker for BRCA1 mutated cancers. This involves the analysis of β‐hCG expression in cancer tissues, followed by a detailed investigation of its link with BRCA1 mutation and analysis of its protein level in the serum of BRCA1 defective cancer patients also, to understand the mechanism of transcriptional regulation of BRCA1 over β‐hCG.MethodsImmunohistochemical analysis, ELISA, Cloning, Protein expression and purification, Electrophoretic Mobility shift Assay (EMSA)ResultsImmunohistochemical analysis of β‐hCG in tumor tissue microarray showed a strong positive expression of β‐hCG in BRCA1mutated breast cancer. Significant levels of β‐hCG in serum was not observed in breast cancer patients when compared to age matched normal control group probably because detectable levels of β‐hCG may not be present in blood. This analysis has to be carried out in a larger cohort of patient samples. To understand the transcriptional regulation of BRCA1 over β‐hCG, the DNA binding domains of BRCA1were cloned into pET‐28b vector, expressed in BL21DE3 cells and purified by Ni‐NTA affinity chromatography. Purified proteins were used for electrophoretic mobility shift assay (EMSA). Results of EMSA showed that BRCA1 did not exhibit any specific binding with β‐hCG promoter. These results suggest that BRCA1 regulation could be due to protein‐protein association with additional DNA binding transcription factors which has to be elucidated in future experiments.ConclusionThe present study highlights the significance of the regulation of β‐hCG in BRCA1 mutated cancers, since a very high expression of β‐hCG in BRCA1 mutated breast cancer is observed, further a detailed study on the same will provide more insights into the exact role of this molecule during breast cancer progression.

Coactivator p300-induced H3K27 acetylation mediates lipopolysaccharide-induced inflammatory mediator synthesis

To investigate the role of acetylated modification induced by coactivator p300 in lipopolysaccharide (LPS)- induced inflammatory mediator synthesis and its molecular mechanism. Agilent SurePrint G3 Mouse Gene Expression V2 microarray chip and Western blotting were used to screen the molecules whose expression levels in mouse macrophages (RAW246.7) were correlated with the stimulation intensity of LPS. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (chip-qPCR) were used to verify the binding of the molecules to the promoters of IL-6 and TNF-α genes. The effects of transfection of RAW246.7 cells with overexpression or interfering plasmids on IL-6 and TNF-α synthesis were evaluated with ELISA, and the binding level of the target molecules and acetylation level of H3K27 in the promoter region of IL-6 and TNF-α genes were analyzed by chromatin immunoprecipitation sequencing technique (chip-seq). Gene microarray chip data and Western blotting both confirmed a strong correlation of p300 expression with the stimulation intensity of LPS. Immunocoprecipitation confirmed the binding between p300 and c-myb. The results of EMSA demonstrated that c-myb (P < 0.05), but not p300, could directly bind to the promoter region of IL-6 and TNF-α genes; p300 could bind to the promoters only in the presence of c-myb (P < 0.05). The expressions of p65, p300 and c-myb did not show interactions. Both p300 overexpression and LPS stimulation could increase the level of promoter-binding p300 and H3K27 acetylation level, thus promoting p65 binding and inflammatory gene transcription; such effects were obviously suppressed by interference of c-myb expression (P < 0.05). Interference of p65 resulted in inhibition of p65 binding to the promoters and gene transcription (P < 0.05) without affecting p300 binding or H3K27 acetylation level. LPS can stimulate the synthesis of p300, whose binding to the promoter region of inflammatory genes via c-myb facilitates the cohesion of p65 by inducing H3K27 acetylation, thus promoting the expression of the inflammatory genes.

Identification and functional analysis of variations in ENG promoter from patients of isolated ventricular septal defects associated with pulmonary hypertension

Objective Ventricular septal defect (VSD) is one of the most common types of congenital heart disease (CHD) and pulmonary hypertension (PAH) is a serious complication of VSD. As a transmembrane glycoprotein, Endoglin (ENG) is mainly expressed in endothelial cells, and plays a crucial role in development of cardiopulmonary diseases. Studies have shown that mutations in ENG coding sequence can lead to PAH, but the dysfunction of ENG promoter and its correlation with formation of VSD-PAH are still unclear. We hypothesized that variations in ENG promoter may be related to the pathogenesis of VSD-PAH. Methods Blood samples from 362 patients with isolated VSD, including 184 with PAH and 178 without PAH as control were studied. Total DNA from all subjects was extracted and the PCR combined with Sanger sequencing was used to identify variations from ENG promoter. The luciferase activities of pGL3 basic-ENG promoter with/without variation were detected by dual-luciferase assays at the cellular level. Electrophoretic mobility shift assay (EMSA) was used to test the effect of variations in ENG promoter on binding sites of transcription factors. Results Five novel heterozygous variations were identified in ENG promoter from 4 VSD-PAH patients and 2 from VSD patients. Compared with the wild type, the transcriptional activities of ENG promoter with variation were significantly changed, either up-regulated (g.127854409 C>A, g.127854342C>T, g.127854480 C>T, g.127854697 G>T) or down-regulated (g.127854345 G>A). The results of EMSA showed that variations in ENG promoter affected the number and affinity of binding sites of transcription factors. Conclusion Variations identified from ENG promoter sequence in VSD-PAH patients affect the transcriptional activity and therefore are possibly involved in the development of PAH. These findings may provide a new perspective into the molecular pathogenesis and potential therapeutic insights for patients with VSD-PAH.

A preliminary computational outputs versus experimental results: Application of sTRAP, a biophysical tool for the analysis of SNPs of transcription factor-binding sites.

BackgroundIn the human genome, the transcription factors (TFs) and transcription factor‐binding sites (TFBSs) network has a great regulatory function in the biological pathways. Such crosstalk might be affected by the single‐nucleotide polymorphisms (SNPs), which could create or disrupt a TFBS, leading to either a disease or a phenotypic defect. Many computational resources have been introduced to predict the TFs binding variations due to SNPs inside TFBSs, sTRAP being one of them.MethodsA literature review was performed and the experimental data for 18 TFBSs located in 12 genes was provided. The sequences of TFBS motifs were extracted using two different strategies; in the size similar with synthetic target sites used in the experimental techniques, and with 60 bp upstream and downstream of the SNPs. The sTRAP (http://trap.molgen.mpg.de/cgi-bin/trap_two_seq_form.cgi) was applied to compute the binding affinity scores of their cognate TFs in the context of reference and mutant sequences of TFBSs. The alternative bioinformatics model used in this study was regulatory analysis of variation in enhancers (RAVEN; http://www.cisreg.ca/cgi-bin/RAVEN/a). The bioinformatics outputs of our study were compared with experimental data, electrophoretic mobility shift assay (EMSA).ResultsIn 6 out of 18 TFBSs in the following genes COL1A1, Hb ḉᴪ, TF, FIX, MBL2, NOS2A, the outputs of sTRAP were inconsistent with the results of EMSA. Furthermore, no p value of the difference between the two scores of binding affinity under the wild and mutant conditions of TFBSs was presented. Nor, were any criteria for preference or selection of any of the measurements of different matrices used for the same analysis.ConclusionOur preliminary study indicated some paradoxical results between sTRAP and experimental data. However, to link the data of sTRAP to the biological functions, its optimization via experimental procedures with the integration of expanded data and applying several other bioinformatics tools might be required.

Ethanol extract of Elephantopus scaber Linn. Attenuates inflammatory response via the inhibition of NF-κB signaling by dampening p65-DNA binding activity in lipopolysaccharide-activated macrophages.

Elephantopus scaber Linn. (E.scaber) is a widely-used traditional herb whose use has been documented for various inflammatory diseases such as fever, sore throat, dysentery, carbuncle and so on. However, the effect and mechanism of E.scaber in LPS-activated macrophages remain unclear. This study aims to investigate the anti-inflammatory mechanism of the ethanol extract of E.scaber (ESE) in lipopolysaccharide (LPS)-induced inflammatory models. Griess reagent was used to determine NO production, and the levels of TNF-α, IL-6, MCP-1 and IL-1β were determined by ELISA kits. The molecular mechanism research was performed by RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA). LPS-induced endotoxemia mouse model was used for evaluating the in vivo anti-inflammatory action of ESE. ESE suppressed LPS-induced iNOS, TNF-α, IL-6, MCP-1 and IL-1β transcription as well as supernatant NO, TNF-α, IL-6, MCP-1 and IL-1β production in macrophages. Although ESE inhibited NF-κB activation, it did not affect the IκBα phosphorylation and degradation and the NF-κB p65 nuclear translocation. The result of EMSA revealed that ESE inhibited the NF-κB p65-DNA binding activity. Additionally, ESE also decreased the proinflammatory cytokines in serum and peritoneal lavage fluid of LPS-induced endotoxemic mice. ESE has a potently anti-inflammatory effect through inhibiting the NF-κB p65-DNA binding activity in LPS-activated macrophages.

Lead Neurotoxicity on Human Neuroblastoma Cell Line SH-SY5Y is Mediated via Transcription Factor EGR1/Zif268 Induced Disrupted in Scherophernia-1 Activation.

Lead (Pb2+) is a well-known type of neurotoxin and chronic exposure to Pb2+ induces cognition dysfunction. In this work, the potential role of early growth response gene 1 (EGR1) in the linkage of Pb2+ exposure and disrupted in scherophernia-1 (DISC1) activity was investigated. Human neuroblastoma cell line SH-SY5Y was subjected to different concentrations of lead acetate (PbAc) to determine the effect of Pb2+ exposure on the cell viability, apoptosis, and activity of EGR1 and DISC1. Then the expression of EGR1 in SH-SY5Y cells was knocked down with specific siRNA to assess the function of EGR1 in Pb2+ induced activation of DISC1. The interaction between EGR1 and DISC1 was further validated with dual luciferase assay, Supershift electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP)-PCR. Administration of PbAc decreased cell viability and induced apoptosis in SH-SY5Y cells in a dose-dependent manner. Additionally, exposure to PbAc also up-regulated expression of EGR1 and DISC1 at all concentrations. Knockdown of EGR1 blocked the effect of PbAc on SH-SY5Y cells, indicating the central role of EGR1 in the function of Pb2+ on activity of DISC1. Based on the results of dual luciferase assay, Supershift EMSA, and ChIP-PCR, EGR1 mediated the effect of Pb2+ on DISC1 by directly bound to the promoter region of DISC1 gene. The current study elaborated the mechanism involved in the effect of Pb2+ exposure on expression of DISC1 for the first time: EGR1 activated by Pb2+ substitution of zinc triggered the transcription of DISC1 gene by directly binding to its promoter.

Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance.

Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish in-vitro culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-β proteins (4) to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-κB to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11–7082 treatment (5μM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF-κB to Hb-α promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-α and Hb-β and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-κB regulatory sequences in Hb-α promoter, whose activation is associated with immune response of hPVECs, indicating Hb-α and Hb-β may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.

Abstract 3373: TGF-β1 signaling mediated lymphangiogenesis in gastric cancer

Abstract Background Recent evidence showed that Transforming growth factor (TGF)-β1 could have important role for gastric cancer progression and metastasis. However, the role of TGF- β1 for lymph node metastasis and lymphangiogenesis, the important steps of gastric cancer progression, is largely unknown. To investigate the role of TGF-β1 on lymphangiogenesis and its molecular mechanisms is the main purpose of this study. Methods We selected two gastric cancer cell-line models, MKN45 and KATOIII, which hold appropriate characteristics for the current study. We checked the possibility of the autocrine function of TGF-β1 for lymphangiogenesis in these gastric cancer cell-lines. We investigated the changes of TGF-β1 signaling molecules and VEGF-C expression in responding to TGF-β1 and its inhibitor. To examine the binding of Smad3 to the promoter region of VEGF-C, we performed electrophoretic mobility shift assay (EMSA). Both Smad-dependent and Smad-independent pathways were also examined to elucidate which pathways were involved in lymphangiogenesis. Finally, tube forming assay was performed to validate phenotypical effect of TGF-β1 on lymphangiogenesis. Results Two gastric cancer cell-line models, MKN45 and KATOIII, showed autocrine regulation of TGF-β1. TGF-β1 signaling was transduced to VEGF-C, mediated by Smad3. Smad3 was bound to the promoter of VEGF-C which was confirmed with EMSA. Based on EMSA results, KATOIII showed stronger reaction between Smad3 and VEGF-C promoter than MKN45 did. In MKN45, TGF-β1 also transduced its signal to VEGF-C through PI3k-Akt pathway, one of the Smad-independent pathway. Lymphatic endothelial cells cultured in the condition media of MKN45 and KATOIII underwent tubule structure formation. The growth of HLEC was accelerated with TGF-β1 mixed conditioned media, while failed to induce tubular structure in the presence of TGF-β1 inhibitor. The expression level of secreted VEGF-C was as same in responding to TGF-β1 and its inhibitor as the lymphatic tube forming results in gastric cancer cells. Conclusion TGF-β1 could promote lymphangiogenesis through VEGF-C expression in gastric cancer cell line models. TGF-β1 signaling could be transduced through either Smad-mediated or non- Smad-mediated depending on gastric cancer cell-line characteristics. Citation Format: Kyung Ho Pak, Jae-Ho Cheong, Ki Chung Park. TGF-β1 signaling mediated lymphangiogenesis in gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3373.

Valproate ameliorates nitroglycerin-induced migraine in trigeminal nucleus caudalis in rats through inhibition of NF-кB.

BackgroundAs a complex nervous system disease, migraine causes severe healthy and social issues worldwide. Valproate (VPA) is a widely used treatment agent against seizures and bipolar disorder, and its function to alleviate damage due to migraine has also been verified in clinical investigations. However, the mechanism underlying the protective effect of VPA against migraine remains poorly revealed. In the current study, the major purpose was to uncover the mechanism which drove VPA to antagonize migraine.MethodsNitroglycerin (NTG) was employed to induce a migraine model in rats and the migraine animals were exposed to treatment of VPA of different doses. Thereafter, the levels of indicators related to oxidative stress were measured and used to evaluate the anti-oxidant potential of VPA. The expression of calcitonin gene-related peptide (CGRP) and c-Fos was also quantified with ELISA and immunohistochemistry, respectively. Western blotting and electrophoretic mobility shift assays (EMSA) were conducted to explore the effect of VPA treatment on NF-кB pathway.ResultsNTG induced the activation of oxidative stress and led to migraine in model animals, but pre-treatment with VPA attenuated the damage due to migraine attack in brain tissues. The level of lipid peroxidation was significantly reduced while the prodcution of anti-oxidant factors was restored. Furthermore, expressions of CGRP and c-Fos, which represented the neuronal activation, were also down-regulated by VPA. The results of western blotting and EMSA demonstrated that the above mentioned effect of VPA acted through the inhibition of NF-кB pathway.ConclusionsAlthough controversies on the effect of VPA on NF-кB pathway existed, our study revealed an alternative mechanism of VPA in protecting against migraine, which would promote the development of therapeutic strategies of migraine.

Hoogsteen-position pyrimidines promote the stability and function of the MALAT1 RNA triple helix.

Triple-stranded RNA was first deduced to form in vitro more than 50 years ago and has since been implicated in RNA catalysis, stability, and small molecule binding. Despite the emerging biological significance of RNA triple helices, it remains unclear how their nucleotide composition contributes to their thermodynamic stability and cellular function. To investigate these properties, we used in vitro RNA electrophoretic mobility shift assays (EMSAs) and in vivo intronless β-globin reporter assays to measure the relative contribution of 20 RNA base triples (N•A–U, N•G–C, N•C–G, N•U–A, and N•G–U) to triple-helical stability. These triples replaced a single internal U•A–U within the known structure of the triple-helical RNA stability element of human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which contains 10 major-groove base triples. In addition to the canonical C•G–C triple, the noncanonical base triples U•G–C, U•G–U, C•C–G, and U•C–G exhibited at least 30% stability relative to the wild-type U•A–U base triple in both assays. Of these triples, only U•A–U, C•G–C, and U•G–C, when tested as four successive triples, formed stabilizing structures that allowed accumulation of the intronless β-globin reporter. Overall, we find that Hoogsteen-position pyrimidines support triple helix stability and function and that thermodynamic stability, based on EMSA results, is necessary but not sufficient for stabilization activity of the MALAT1 triple helix in cells. These results suggest that additional RNA triple helices containing noncanonical triples likely exist in nature.

Human Obesity Associated with an Intronic SNP in the Brain-Derived Neurotrophic Factor Locus.

Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

Electroacupuncture alleviates retrieval of pain memory and its effect on phosphorylation of cAMP response element-binding protein in anterior cingulate cortex in rats.

BackgroundRecent evidence suggests that persistent pain and recurrent pain are due to the pain memory which is related to the phosphorylation of cAMP response element-binding protein (p-CREB) in anterior cingulate cortex (ACC). Eletroacupuncture (EA), as a complementary Chinese medical procedure, has a significant impact on the treatment of pain and is now considered as a mind-body therapy.MethodsThe rat model of pain memory was induced by two injections of carrageenan into the paws, which was administered separately by a 14-day interval, and treated with EA therapy. The paw withdrawal thresholds (PWTs) of animals were measured and p-CREB expressions in ACC were detected by using immunofluorescence (IF) and electrophoretic mobility shift assay (EMSA). Statistical comparisons among different groups were made by one-way, repeated-measures analysis of variance (ANOVA).ResultsThe second injection of carrageenan caused the decrease of PWTs in the non-injected hind paw. EA stimulation applied prior to the second injection, increased the values of PWTs. In ACC, the numbers of p-CREB positive cells were significantly increased in pain memory model rats, which were significantly reduced by EA. EMSA results showed EA also down-regulated the combining capacity of p-CREB with its DNA. Furthermore, the co-expression of p-CREB with GFAP, OX-42, or NeuN in ACC was strengthened in the pain memory model rats. EA inhibited the co-expression of p-CREB with GFAP or OX-42, but not NeuN in ACC.ConclusionsThe present results suggest the retrieval of pain memory could be alleviated by the pre-treatment of EA, which is at least partially attributed to the down-regulated expression and combining capacity of p-CREB and the decreased expression of p-CREB in astrocytes and microglia cells.

Regulation of Mcl-1 by constitutive activation of NF-kappaB contributes to cell viability in human esophageal squamous cell carcinoma cells

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with a 5-year survival rate less than 15%. Understanding of the molecular mechanisms involved in the pathogenesis of ESCC becomes critical to develop more effective treatments.MethodsMcl-1 expression was measured by reverse transcription (RT)-PCR and Western blotting. Human Mcl-1 promoter activity was evaluated by reporter gene assay. The interactions between DNA and transcription factors were confirmed by electrophoretic mobility shift assay (EMSA) in vitro and by chromatin immunoprecipitation (ChIP) assay in cells.ResultsFour human ESCC cell lines, TE-1, Eca109, KYSE150 and KYSE510, are revealed increased levels of Mcl-1 mRNA and protein compare with HaCaT, an immortal non-tumorigenic cell line. Results of reporter gene assays demonstrate that human Mcl-1 promoter activity is decreased by mutation of kappaB binding site, specific NF-kappaB inhibitor Bay11-7082 or dominant inhibitory molecule DNMIkappaBalpha in TE-1 and KYSE150 cell lines. Mcl-1 protein level is also attenuated by Bay11-7082 treatment or co-transfection of DNMIkappaBalpha in TE-1 and KYSE150 cells. EMSA results indicate that NF-kappaB subunits p50 and p65 bind to human Mcl-1-kappaB probe in vitro. ChIP assay further confirm p50 and p65 directly bind to human Mcl-1 promoter in intact cells, by which regulates Mcl-1 expression and contributes to the viability of TE-1 cells.ConclusionsOur data provided evidence that one of the mechanisms of Mcl-1 expression in human ESCC is regulated by the activation of NF-kappaB signaling. The newly identified mechanism might provide a scientific basis for developing effective approaches to treatment human ESCC.

Management of Cytopenias With Ruxolitinib Treatment in Patients With Myelofibrosis

Primary myelofibrosis (PMF) is a stem cellederived hematologic malignancy, characterized by an expansion of myeloid lineages resulting in, among other, bone marrow (BM) reticulin and/or collagen fibrosis. An acquired activating mutation in Janus kinase 2 at codon V617F (JAK2V617F) is detected in BM cells of the majority of patients with PMF. Constitutively activated JAK2 induces phosphorylation and activation of STAT3 that upon binding to DNA activates STAT3target genes and induces production of various cytokines. As reported previously, most of these hematopoietic stroma derived soluble factors activate NF-kB that plays an important role in the survival and proliferation of normal and neoplastic cells. The canonical NF-kB activation pathway is typically initiated by extracellular signals that activate the b subunit of the IkB kinase (IKK) complex (IKKb) which induces the phosphorylation and degradation of the NF-kB inhibitor IkBa. Following IkBa degradation, NF-kB heterodimers translocate to the nucleus and bind to DNA. We have recently found that in chronic lymphocytic leukemia (CLL) constitutively activated STAT3 induces the production of unphsophorylated (U) STAT3 which binds to the NF-kB dimers p65/p50 in competition with IkB. U-STAT3/NF-kB complex shuttles to the nucleus where NF-kB binds to DNA and activates NF-kB-regulated genes (Liu et al. Mol Cancer Res 9: 507, 2011). Because in PMF constitutively activated JAK2 induces phosphorylation of STAT3, subsequently leading to the production of U-STAT3, we wondered whether, like in CLL, U-STAT3 also activates NF-kB in PMF. By studying low-density BM cells of 11 untreated patients with PMF using the electrophoretic mobility shift assay (EMSA), we found that NF-kB is constitutively activated in all PMF BM samples. Consequently, we hypothesized that, like in CLL cells, activation of NF-kB in PMF cells is induced by an IkB-unrelated mechanism as reported by Yang J et al. (Cancer Res 65:939, 2005). By using immunoprecipitation we determined that STAT3 binds to the RelA/p65 NF-kB protein whilst results of EMSA showed that anti-STAT3, similar to antiNF-kB p65 antibodies, attenuated the binding of PMF BM cell extract to the NF-kB DNA probe. Taken together, our data suggest that U-STAT3 binds the NFkB dimers p65/p50 and constitutively activates NF-kB in PMF.

KLF5 strengthens drug resistance of ovarian cancer stem‐like cells by regulating survivin expression

Ovarian cancer stem-like cells (CSCs), which can form non-adherent sphere cells in a stem-cell selection culture system, exhibit stemness and drug resistance to chemotherapeutics, which are properties not observed in differentiated cells. Recent studies have demonstrated that Kruppel-like factor 5 (KLF5) is involved in cell proliferation and mediates cell survival and tumourigenesis. Here, we investigated the role of KLF5 and its downstream target survivin, in strengthening drug resistance of ovarian CSCs. Ovarian cancer cell line SKOV3 was cultured under serum-free conditions and differentiating conditions to promote formation of sphere cells and differentiated cells, respectively. siRNA-KLF5 was used to knock down KLF5, and survivin expression vector was used to overexpress survivin. Cells were further analysed by qPCR, immunofluorescence staining and western blotting. Chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) were performed to investigate the relationship between KLF5 and survivin expression. Drug resistance was examined by MTT and apoptosis assays. KLF5 was highly expressed in the ovarian cancer cell line SKOV3 sphere cells, accompanied by elevated survivin expression. Silencing KLF5 by small interfering RNA in sphere cells down-regulated survivin expression, which also sensitized the sphere cells to apoptosis induced by chemotherapeutic drugs (cisplatin or paclitaxel). Furthermore, ChIP assay, survivin overexpression and EMSA results indicated that KLF5 controlled survivin expression by directly binding the surivin promoter in the cells. The KLF5-mediated signalling pathway is a potential target for elimination of ovarian CSCs.

O21.5 Understanding the Molecular Mechanism of mtrR in the Regulation of Antimicrobial Resistance in Neisseria Gonorrhoeae Using in Vitro and In Silico Studies

BackgroundNeisseria gonorrhoeae, a major STD causing pathogens, tends to pose high burden of morbidity that is borne disproportionately by women and infants with approximately 2/3rd of cases from developing countries....

1,25-Dihydroxyvitamin D3 up-regulates expression of hsa-let-7a-2 through the interaction of VDR/VDRE in human lung cancer A549 cells

AimsWe aim to investigate the relationship between 1,25-(OH)2VD3 and hsa-let-7a in lung cancer A549 cells. MethodsReal-time PCR and luciferase reporter assays were used to detect the influence of 1,25-(OH)2VD3 on the expression of hsa-let-7a-2 after A549 cells were treated with 1,25-(OH)2VD3 (10−8~10−6mol/L). Analysis of the 5.0Kb upstream sequence of the pre-let-7a-2 showed that one vitamin D response element (VDRE) is located in -2066/-2042bp of pre-let-7a-2. Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to determine whether 1,25-(OH)2VD3 activating vitamin D receptor (VDR) could bind to this VDRE to promote hsa-let-7a-2 expression. ResultsWe found that 1,25-(OH)2VD3 could up-regulate the expression of hsa-let-7a-2 in a dose-dependent manner. The results of EMSA and ChIP demonstrated that 1,25-(OH)2VD3/VDR could interact with the VDRE in the upstream of pre-let-7a-2. Luciferase reporter assay showed that this VDRE is a functional cis-element mediating the up-regulation of hsa-let-7a-2 expression induced by 1,25-(OH)2VD3. ConclusionsOur data indicated that 1,25-(OH)2VD3 could up-regulate the transcription of hsa-let-7a-2 in lung cancer cells, and the up-regulation of hsa-let-7a-2 expression induced by 1,25-(OH)2VD3 might mediate the anti-proliferation effects of 1,25-(OH)2VD3 in lung cancer cells.

Exendin‐4 inhibits iNOS expression at the protein level in LPS‐stimulated Raw264.7 macrophage by the activation of cAMP/PKA pathway

Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet β-cells. However, GLP-1 receptors are present in many extrapancreatic tissues including macrophages, and thus GLP-1 may have diverse actions in these tissues and cells. Therefore, we examined the mechanism by which exendin-4 (EX-4), a potent GLP-1 receptor agonist, inhibits lipopolysaccharide (LPS)-induced iNOS expression in Raw264.7 macrophage cells. EX-4 significantly inhibited LPS-induced iNOS protein expression and nitrite production. However, Northern blot and promoter analyses demonstrated that EX-4 did not inhibit LPS-induced iNOS mRNA expression and iNOS promoter activity. Electrophoretic mobility shift assay (EMSA) showed that EX-4 did not alter the binding activity of NF-κB to the iNOS promoter. Consistent with the result of EMSA, LPS-induced IκBα phosphorylation and nuclear translocation of p65 were not inhibited by EX-4. Also, actinomycin D chase study and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not affect iNOS mRNA stability. Meanwhile, cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. The EX-4 inhibition of LPS-induced iNOS protein was significantly reversed by adenylate cyclase inhibitors (MDL-12330A and SQ 22536), a PKA inhibitor (H-89) and PKAα gene silencing. These findings suggest that EX-4 inhibited LPS-induced iNOS expression at protein level, but not at transcriptional mechanism of iNOS gene and this inhibitory effect of EX-4 was mainly dependent on cAMP/PKA system.

Response to 'TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1β-induced rat osteoarthritis chondrocytes in vitro'

Sodium ferulate (SF) is a natural component of traditional Chinese herbs. Our previous study shows that SF has a protective effect on osteoarthritis (OA). The objective of this study was to investigate the effect of SF on the TNF/TNF receptor (TNFR) signal transduction pathway of rat OA chondrocytes.Primary rat articular chondrocytes were co-treated with IL-1β and SF. Chondrocyte apoptosis was assessed by fluorescein isothiocyanate-annexin V/propidium iodide assay. The PCR array was used to screen the expression of 84 key genes involved in apoptosis. The release of TNFα and prostaglandin E2 were analyzed by ELISA. Expressions of proteins were assessed by western blotting. The activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Gene expression of inducible nitric oxide synthase (iNOS) was evaluated by real-time quantitative PCR. The nitric oxide content was measured with the Griess method.After treatment with SF, the apoptosis rate of chondrocytes significantly attenuated (P < 0.01). Results of the apoptosis PCR array suggested that mRNA expression of some core proteins in the TNF/TNFR pathway showed valuable regulation. The protein expressions of TNFα, TNFR-1, TNF receptor-associated death domain, caspase-8 and caspase-3 were prevented by SF in a concentration-dependent manner. SF also inhibited activities of caspase-8 and caspase-3 compared with the OA model control (P < 0.01). TNF receptor-associated factor-2 expression, phosphorylations of inhibitor of NF-κB kinase (IKK) subunits alpha and beta, and NF-κB inhibitor, alpha (IκBα) were all concentration-dependently suppressed by SF treatment. The results of EMSA showed that SF inhibited the activity of NF-κB. In addition, the expressions of cycloxygenase-2 and iNOS and the contents of prostaglandin E2 and NO were attenuated with the treatment of SF (P < 0.01).SF has anti-apoptosis and anti-inflammatory effects on an OA model induced by IL-1β in vitro, which were due to inhibitory actions on the caspase-dependent apoptosis pathway and the IKK/NF-κB signal transduction pathway of the TNF/TNFR pathway.