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  • Long non-coding RNA HOTAIR, a c-Myc activated driver of malignancy, negatively regulates miRNA-130a in gallbladder cancer. 24953832

    Protein coding genes account for only about 2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs including long non-coding RNAs. A growing volume of literature has proposed that lncRNAs are important players in cancer. HOTAIR was previously shown to be an oncogene and negative prognostic factor in a variety of cancers. However, the factors that contribute to its upregulation and the interaction between HOTAIR and miRNAs are largely unknown.A computational screen of HOTAIR promoter was conducted to search for transcription-factor-binding sites. HOTAIR promoter activities were examined by luciferase reporter assay. The function of the c-Myc binding site in the HOTAIR promoter region was tested by a promoter assay with nucleotide substitutions in the putative E-box. The association of c-Myc with the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay and Electrophoretic mobility shift assay. A search for miRNAs with complementary base paring with HOTAIR was performed utilizing online software program. Gain and loss of function approaches were employed to investigate the expression changes of HOTAIR or miRNA-130a. The expression levels of HOTAIR, c-Myc and miRNA-130a were examined in 65 matched pairs of gallbladder cancer tissues. The effects of HOTAIR and miRNA-130a on gallbladder cancer cell invasion and proliferation was tested using in vitro cell invasion and flow cytometric assays.We demonstrate that HOTAIR is a direct target of c-Myc through interaction with putative c-Myc target response element (RE) in the upstream region of HOTAIR in gallbladder cancer cells. A positive correlation between c-Myc and HOTAIR mRNA levels was observed in gallbladder cancer tissues. We predicted that HOTAIR harbors a miRNA-130a binding site. Our data showed that this binding site is vital for the regulation of miRNA-130a by HOTAIR. Moreover, a negative correlation between HOTAIR and miRNA-130a was observed in gallbladder cancer tissues. Finally, we demonstrate that the oncogenic activity of HOTAIR is in part through its negative regulation of miRNA-130a.Together, these results suggest that HOTAIR is a c-Myc-activated driver of malignancy, which acts in part through repression of miRNA-130a.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • A NF-κB-dependent dual promoter-enhancer initiates the lipopolysaccharide-mediated transcriptional activation of the chicken lysozyme in macrophages. 23533622

    The transcriptional activation of the chicken lysozyme gene (cLys) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF-κB, AP1 and C/EBPβ controls this CRE, which is strictly dependent on NF-κB binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKKα, known to be recruited by NF-κB to NF-κB-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF-κB binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKKα detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3' end of the gene.
    Document Type:
    Reference
    Product Catalog Number:
    05-690
    Product Catalog Name:
    Anti-HP1γ Antibody, clone 42s2

  • Capsaicin suppresses the migration of cholangiocarcinoma cells by down-regulating matrix metalloproteinase-9 expression via the AMPK-NF-κB signaling pathway. 25217963

    Cholangiocarcinoma is one of the most difficult malignancies to cure. An important prognostic factor is metastasis, which precludes curative surgical resection. Recent evidence shows that capsaicin has an inhibitory effect on cancer cell migration and invasion. Here, we investigated the molecular mechanism of the capsaicin-induced anti-migration and anti-invasion effects on HuCCT1 cholangiocarcinoma cells. Migration and invasion were significantly reduced in response to capsaicin. Capsaicin also inhibited the expression of matrix metalloproteinase-9 (MMP-9). In capsaicin-treated cells, levels of phosphorylated AMPK increased, and this effect was abolished by treatment with the AMPK inhibitor, Compound C. Capsaicin enhanced the expression of SIRT1, which can activate the transcription factor NF-κB by deacetylation. This suggests that NF-κB is activated by capsaicin via the SIRT1 pathway. In addition, capsaicin-activated AMPK induced the phosphorylation of IκBα and nuclear localization of NF-κB p65. Chromatin immunoprecipitation assays demonstrated that capsaicin reduced MMP-9 transcription by inhibiting NF-κB p65 translocation and deacetylation via SIRT1. These findings provide evidence that capsaicin suppresses the migration and invasion of cholangiocarcinoma cells by inhibiting NF-κB p65 via the AMPK-SIRT1 and the AMPK-IκBα signaling pathways, leading to subsequent suppression of MMP-9 expression.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • The natural chemopreventive agent sulforaphane inhibits STAT5 activity. 24910998

    Signal transducer and activator of transcription STAT5 is an essential mediator of cytokine, growth factor and hormone signaling. While its activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated to a number of hematological and solid tumor cancers. We previously showed that deacetylase inhibitors can inhibit STAT5 transcriptional activity. We now investigated whether the dietary chemopreventive agent sulforaphane, known for its activity as deacetylase inhibitor, might also inhibit STAT5 activity and thus could act as a chemopreventive agent in STAT5-associated cancers. We describe here sulforaphane (SFN) as a novel STAT5 inhibitor. We showed that SFN, like the deacetylase inhibitor trichostatin A (TSA), can inhibit expression of STAT5 target genes in the B cell line Ba/F3, as well as in its transformed counterpart Ba/F3-1*6 and in the human leukemic cell line K562 both of which express a constitutively active form of STAT5. Similarly to TSA, SFN does not alter STAT5 initial activation by phosphorylation or binding to the promoter of specific target genes, in favor of a downstream transcriptional inhibitory effect. Chromatin immunoprecipitation assays revealed that, in contrast to TSA however, SFN only partially impaired the recruitment of RNA polymerase II at STAT5 target genes and did not alter histone H3 and H4 acetylation, suggesting an inhibitory mechanism distinct from that of TSA. Altogether, our data revealed that the natural compound sulforaphane can inhibit STAT5 downstream activity, and as such represents an attractive cancer chemoprotective agent targeting the STAT5 signaling pathway.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Identification of EGF-NF-κB-FOXC1 signaling axis in basal-like breast cancer. 28629477

    The pathogenesis of human basal-like breast cancer (BLBC) is not well understood and patients with BLBC have a poor prognosis. Expression of the epidermal growth factor receptor (EGFR) and nuclear factor-κB (NF-κB) is well-known to be upregulated in BLBC. The forkhead box C1 (FOXC1) transcription factor, an important prognostic biomarker specific for BLBC, has been shown to be induced by EGF and is critical for EGF effects in breast cancer cells. How FOXC1 is transcriptionally activated in BLBC is not clear.Luciferase reporter assays were performed to show that NF-κB-p65 enhances FOXC1 promoter activity in BLBC cells (MDA-MB-468). Electrophoretic mobility shift assay, biotinylated oligonucleotide precipitation assay, and chromatin immunoprecipitation assay were used to show that NF-κB interacts and binds to the promoter region of FOXC1.In this study, we demonstrate that NF-κB is a pivotal mediator of the EGF/EGFR regulation of FOXC1 expression by binding to the FOXC1 promoter to activate FOXC1 transcription. Loss or inhibition of NF-κB diminished FOXC1 expression.Collectively, our findings reveal a novel EGFR-NF-κB-FOXC1 signaling axis that is critical for BLBC cell function, supporting the notion that intervention in the FOXC1 pathway may provide potential modalities for BLBC treatment.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Regulation of the PMP22 gene through an intronic enhancer. 21411665

    Successful myelination of the peripheral nervous system depends upon induction of major protein components of myelin, such as peripheral myelin protein 22 (PMP22). Myelin stability is also sensitive to levels of PMP22, as a 1.4 Mb duplication on human chromosome 17, resulting in three copies of PMP22, is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth disease. The transcription factor Egr2/Krox20 is required for induction of high level expression of Pmp22 in Schwann cells but its activation elements have not yet been determined. Using chromatin immunoprecipitation analysis of the rat Pmp22 locus, we found a major peak of Egr2 binding within the large intron of the Pmp22 gene. Analysis of a 250 bp region within the largest intron showed that it is strongly activated by Egr2 expression in reporter assays. Moreover, this region contains conserved binding sites not only for Egr2 but also for Sox10, which is also required for Schwann cell development. Our analysis shows that Sox10 is required for optimal activity of the intronic site as well as PMP22 expression. Finally, mouse transgenic analysis revealed tissue-specific expression of this intronic sequence in peripheral nerve. Overall, these data show that Egr2 and Sox10 activity are directly involved in mediating the developmental induction of Pmp22 expression.
    Document Type:
    Reference
    Product Catalog Number:
    12-370
    Product Catalog Name:
    Normal Rabbit IgG

  • Differential expression of matrix metalloproteinases in brain- and bone-seeking clones of metastatic MDA-MB-231 breast cancer cells. 16850107

    Matrix Metalloproteinases (MMPs) play a crucial role in breast cancer metastasis. We examined the mRNA and protein expression of several MMPs in brain- and bone-seeking clones of MDA-MB-231 breast cancer cells, their transcriptional regulation and their functional role in the metastatic process. MMP mRNA expression was examined using real-time reverse transcription polymerase chain reaction. Protein expression was examined using enzyme linked immunosorbent essay (ELISA). The inducibility of mRNA and protein expression was tested with TPA (phorbol 12-myristate 13-acetate; 50 microM); epidermal growth factor and transforming growth factor beta (20 ng/ml both). Migration and invasion assays were performed with the QCM 96-Well Migration/Invasion Assay (8 microm; Chemicon) over 24 h with or without specific MMPs inhibitors (MMP Inhibitor I Mix (5 microM); MMP-2/MMP-9 Inhibitor III (50 microM); EMD Biosciences). We found significantly higher mRNA expression of MMP-1 and -9 in brain-seeking 231-clones in comparison to -bone and -parental cells. In contrast, the mRNA expression of MMP-3 and -14 was comparable in all cells lines examined and MMP-13 expression was lower in both selective metastatic lines. MMP-2 and -8 were not expressed. ELISA revealed a higher amount of total as well as active MMP-1 and -9 in brain-seeking cells. TPA stimulation showed that MMP-1 and -9 transcription was inducible on the mRNA and protein level in 231-parental but not in 231-brain or -bone. 231-brain showed the highest migration and invasive capacity which could be decreased by the application of MMP-1 and/or MMP-9 inhibitor. Our results indicate functional importance of MMP-1 and -9 overexpression in brain metastasis in an in vitro model.
    Document Type:
    Reference
    Product Catalog Number:
    ECM555
    Product Catalog Name:
    QCM ECMatrix Cell Invasion Assay, 96-well (8 µm), fluorimetric

  • Phosphorylation of steroidogenic factor 1 is mediated by cyclin-dependent kinase 7. 17901130

    The nuclear receptor steroidogenic factor-1 (SF1) is critical for development and function of steroidogenic tissues. Posttranslational modifications are known to influence the transcriptional capacity of SF1, and it was previously demonstrated that serine 203 is phosphorylated. In this paper we report that serine 203 is phosphorylated by a cyclin-dependent kinase 7 (CDK7)-mediated process. As part of the CDK-activating kinase complex, CDK7 is a component of the basal transcription factor TFIIH, and phosphorylation of SF1 as well as SF1-dependent transcription was clearly reduced in cells carrying a mutation that renders the CDK-activating kinase complex unable to interact with the TFIIH core. Coimmunoprecipitation analyses revealed that SF1 and CDK7 reside in the same complex, and kinase assays demonstrated that immunoprecipitated CDK7 and purified TFIIH phosphorylate SF1 in vitro. The CDK inhibitor roscovitine blocked phosphorylation of SF1, and an inactive form of CDK7 repressed the phosphorylation level and the transactivation capacity of SF1. Structural studies have identified phosphoinositides as potential ligands for SF1. Interestingly, we found that mutations designed to block phospholipid binding dramatically decreased the level of SF1 phosphorylation. Together our results suggest a connection between ligand occupation and phosphorylation and association with the basic transcriptional machinery, indicating an intricate regulation of SF1 transactivation.
    Document Type:
    Reference
    Product Catalog Number:
    07-618
    Product Catalog Name:
    Anti-SF-1 Antibody

  • Interferon-microRNA signalling drives liver precancerous lesion formation and hepatocarcinogenesis. 26860770

    Precancerous lesion, a well-established histopathologically premalignant tissue with the highest risk for tumourigenesis, develops preferentially from activation of DNA damage checkpoint and persistent inflammation. However, little is known about the mechanisms by which precancerous lesions are initiated and their physiological significance.Laser capture microdissection was used to acquire matched normal liver, precancerous lesion and tumour tissues. miR-484(-/-), Ifnar1(-/-) and Tgfbr2(△hep) mice were employed to determine the critical role of the interferon (IFN)-microRNA pathway in precancerous lesion formation and tumourigenesis. RNA immunoprecipitation (RIP), pull-down and chromatin immunoprecipitation (ChIP) assays were applied to explore the underlying mechanisms.miR-484 is highly expressed in over 88% liver samples clinically. DEN-induced precancerous lesions and hepatocellular carcinoma were dramatically impaired in miR-484(-/-) mice. Mechanistically, ectopic expression of miR-484 initiates tumourigenesis and cell malignant transformation through synergistic activation of the transforming growth factor-β/Gli and nuclear factor-κB/type I IFN pathways. Specific acetylation of H3K27 is indispensable for basal IFN-induced continuous transcription of miR-484 and cell transformation. Convincingly, formation of precancerous lesions were significantly attenuated in both Tgfbr2(△hep) and Ifnar1(-/-) mice.These findings demonstrate a new protumourigenic axis involving type I IFN-microRNA signalling, providing a potential therapeutic strategy to manipulate or reverse liver precancerous lesions and tumourigenesis.
    Document Type:
    Reference
    Product Catalog Number:
    17-701
    Product Catalog Name:
    EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit

  • Proto-oncogenic miR-744 is upregulated by transcription factor c-Jun via a promoter activation mechanism. 27533465

    Upregulation of miR-744 is associated with poor prognosis in many types of cancer patients, but it is still unclear how miR-744 becomes elevated in these tumors. In this study, we found that ectopic c-Jun elevated miR-744 expression, whereas c-Jun attenuation reduced miR-744 expression. Chromatin immunoprecipitation assay confirmed the direct binding of c-Jun to the promoter of miR-744. The binding site of -343 to -349 bp within the most potential promoter like sequence of miR-744 was further validated by luciferase reporter gene assays. C-Jun-induced miR-744 upregulation could significantly promote migration and invasion of nasopharyngeal carcinoma cells and non-small cell lung cancer (NSCLC) cells, hence ectopic c-Jun was sufficient to rescue the migratory and invasive ability of these cells when miR-744 was knockdown. Additionally, a positive correlation between the expression levels of miR-744 and c-Jun was revealed in NSCLC samples with high (top 10%) level of miR-744 expression from the TCGA dataset. Taken together, our results demonstrated for the first time the regulatory mechanism of miR-744 transcription by c-Jun, providing a potential mechanism underlying the upregulation of miR-744 in cancers.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™