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1.
Sci Rep ; 8(1): 16079, 2018 Oct 25.
Article in English | MEDLINE | ID: mdl-30356171

ABSTRACT

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

2.
Sci Rep ; 7(1): 11772, 2017 09 18.
Article in English | MEDLINE | ID: mdl-28924147

ABSTRACT

The transcription factor NF-κB is constitutively activated in many epithelial tumors but few NF-κB inhibitors are suitable for cancer therapy because of its broad biological effects. We previously reported that the d4-family proteins (DPF1, DPF2, DPF3a/b) function as adaptor proteins linking NF-κB with the SWI/SNF complex. Here, using epithelial tumor cell lines, A549 and HeLaS3, we demonstrate that exogenous expression of the highly-conserved N-terminal 84-amino acid region (designated "CT1") of either DPF2 or DPF3a/b has stronger inhibitory effects on anchorage-independent growth than the single knockdown of any d4-family protein. This indicates that CT1 can function as an efficient dominant-negative mutant of the entire d4-family proteins. By in situ proximity ligation assay, CT1 was found to retain full adaptor function, indicating that the C-terminal region of d4-family proteins lacking in CT1 would include essential domains for SWI/SNF-dependent NF-κB activation. Microarray analysis revealed that CT1 suppresses only a portion of the NF-κB target genes, including representative SWI/SNF-dependent genes. Among these genes, IL6 was shown to strongly contribute to anchorage-independent growth. Finally, exogenous CT1 expression efficiently suppressed tumor formation in a mouse xenograft model, suggesting that the d4-family proteins are promising cancer therapy targets.


Subject(s)
Gene Expression Regulation, Neoplastic , NF-kappa B/metabolism , Neoplasm Proteins/metabolism , Neoplasms/metabolism , A549 Cells , Animals , Chromosomal Proteins, Non-Histone , HeLa Cells , Humans , Mice , Mice, Nude , NF-kappa B/genetics , Neoplasm Proteins/genetics , Neoplasms/genetics , Neoplasms/pathology , Transcription Factors
3.
Sci Rep ; 7(1): 6650, 2017 07 27.
Article in English | MEDLINE | ID: mdl-28751779

ABSTRACT

Because several studies have shown that exogenous miR-199a has antiviral effects against various viruses, including herpesviruses, we examined how miR-199a exerts its antiviral effects using epithelial tumour cell lines infected with herpes simplex virus-1 (HSV-1). We found that both miR-199a-5p and -3p impair the secondary envelopment of HSV-1 by suppressing their common target, ARHGAP21, a Golgi-localized GTPase-activating protein for Cdc42. We further found that the trans-cisternae of the Golgi apparatus are a potential membrane compartment for secondary envelopment. Exogenous expression of either pre-miR-199a or sh-ARHGAP21 exhibited shared phenotypes i.e. alteration of Golgi function in uninfected cells, inhibition of HSV-1 secondary envelopment, and reduction of trans-Golgi proteins upon HSV-1 infection. A constitutively active form of Cdc42 also inhibited HSV-1 secondary envelopment. Endogenous levels of miR-199a in epithelial tumour cell lines were negatively correlated with the efficiency of HSV-1 secondary envelopment within these cells. These results suggest that miR-199a is a crucial regulator of Cdc42 activity on Golgi membranes, which is important for the maintenance of Golgi function and for the secondary envelopment of HSV-1 upon its infection.


Subject(s)
Epithelial Cells/metabolism , GTPase-Activating Proteins/metabolism , Herpes Simplex/metabolism , MicroRNAs/metabolism , cdc42 GTP-Binding Protein/genetics , Cell Line, Tumor , Down-Regulation , Epithelial Cells/virology , Gene Expression Regulation , Golgi Apparatus/metabolism , Herpes Simplex/genetics , Herpesvirus 1, Human/physiology , Humans , Signal Transduction
4.
Sci Rep ; 7(1): 889, 2017 04 18.
Article in English | MEDLINE | ID: mdl-28420882

ABSTRACT

Glioma initiating cells (GICs) are thought to contribute to therapeutic resistance and tumor recurrence in glioblastoma, a lethal primary brain tumor in adults. Although the stem-like properties of GICs, such as self-renewal and tumorigenicity, are epigenetically regulated, the role of a major chromatin remodeling complex in human, the SWI/SNF complex, remains unknown in these cells. We here demonstrate that the SWI/SNF core complex, that is associated with a unique corepressor complex through the d4-family proteins, DPF1 or DPF3a, plays essential roles in stemness maintenance in GICs. The serum-induced differentiation of GICs downregulated the endogenous expression of DPF1 and DPF3a, and the shRNA-mediated knockdown of each gene reduced both sphere-forming ability and tumor-forming activity in a mouse xenograft model. Rescue experiments revealed that DPF1 has dominant effects over DPF3a. Notably, whereas we have previously reported that d4-family members can function as adaptor proteins between the SWI/SNF complex and NF-κB dimers, this does not significantly contribute to maintaining the stemness properties of GICs. Instead, these proteins were found to link a corepressor complex containing the nuclear receptor, TLX, and LSD1/RCOR2 with the SWI/SNF core complex. Collectively, our results indicate that DPF1 and DPF3a are potential therapeutic targets for glioblastoma.


Subject(s)
Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/metabolism , Glioma/genetics , Neoplastic Stem Cells/metabolism , Transcription Factors/metabolism , Cells, Cultured , Co-Repressor Proteins/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Glioma/metabolism , Glioma/pathology , Humans , NF-kappa B/metabolism , Nerve Tissue Proteins/metabolism , Orphan Nuclear Receptors , Receptors, Cytoplasmic and Nuclear/metabolism
5.
Sci Rep ; 6: 21117, 2016 Feb 18.
Article in English | MEDLINE | ID: mdl-26887353

ABSTRACT

Whereas miR-200 family is known to be involved in the epithelial-to-mesenchymal transition (EMT), a crucial biological process observed in normal and pathological contexts, it has been largely unclear how far the functional levels of these tiny RNAs alone can propagate the molecular events to accomplish this process within several days. By developing a potent inhibitor of miR-200 family members (TuD-141/200c), the expression of which is strictly regulatable by the Tet (tetracycline)-On system, we found using a human colorectal cell line, HCT116, that several direct gene target mRNAs (Zeb1/Zeb2, ESRP1, FN1and FHOD1) of miR-200 family were elevated with distinct kinetics. Prompt induction of the transcriptional suppressors, Zeb1/Zeb2 in turn reduced the expression levels of miR-200c/-141 locus, EpCAM, ESRP1 and E-Cad. The loss of ESRP1 subsequently switched the splicing isoforms of CD44 and p120 catenin mRNAs to mesenchymal type. Importantly, within 9 days after the release from the inhibition of miR-200 family, all of the expression changes in the 14 genes observed in this study returned to their original levels in the epithelial cells. This suggests that the inherent epithelial plasticity is supported by a weak retention of key regulatory gene expression in either the epithelial or mesenchymal states through epigenetic regulation.


Subject(s)
Epigenesis, Genetic , Epithelial-Mesenchymal Transition , MicroRNAs/antagonists & inhibitors , Cell Line, Tumor , Epigenesis, Genetic/drug effects , Epigenesis, Genetic/genetics , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Humans , MicroRNAs/biosynthesis , MicroRNAs/genetics
7.
Sci Rep ; 5: 8428, 2015 Feb 12.
Article in English | MEDLINE | ID: mdl-25673149

ABSTRACT

In epithelial cells, miRNA-199a-5p/-3p and Brm, a catalytic subunit of the SWI/SNF complex were previously shown to form a double-negative feedback loop through EGR1, by which human cancer cell lines tend to fall into either of the steady states, types 1 [miR-199a(-)/Brm(+)/EGR1(-)] and 2 [miR-199a(+)/Brm (-)/EGR1(+)]. We show here, that type 2 cells, unlike type 1, failed to form colonies in soft agar, and that CD44, MET, CAV1 and CAV2 (miR-199a targets), all of which function as plasma membrane sensors and can co-localize in caveolae, are expressed specifically in type 1 cells. Single knockdown of any of them suppressed anchorage-independent growth of type 1 cells, indicating that the miR-199a/Brm/EGR1 axis is a determinant of anchorage-independent growth. Importantly, two coherent feedforward loops are integrated into this axis, supporting the robustness of type 1-specific gene expression and exemplifying how the miRNA-target gene relationship can be stably sustained in a variety of epithelial tumors.


Subject(s)
Carcinoma/genetics , Early Growth Response Protein 1/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Transcription Factors/genetics , Carcinoma/metabolism , Carcinoma/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Caveolin 1/genetics , Caveolin 1/metabolism , Caveolin 2/genetics , Caveolin 2/metabolism , Cell Line, Tumor , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Models, Biological , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism
9.
FEBS Lett ; 588(9): 1630-6, 2014 May 02.
Article in English | MEDLINE | ID: mdl-24607481

ABSTRACT

In this study, we demonstrate that the 7SK small nuclear ribonucleoprotein (snRNP) complex is recruited to the HIV-1 promoter via newly-synthesized HIV-1 nascent transcripts (short transcripts) in an hnRNP A1-dependent manner and negatively regulates viral transcript elongation. Our deep-sequence analysis showed these short transcripts were mainly arrested at approximately +50 to +70 nucleotides from the transcriptional start site in the U1 cells, an HIV-1 latent model. TNF-α treatment promptly disrupted the 7SK snRNP complex on the nascent transcripts and viral elongated transcripts were increased. This report provides insight into how 7SK snRNP complex is recruited to HIV-1 promoter in the absence of Tat.


Subject(s)
HIV-1/genetics , RNA, Viral/genetics , Ribonucleoproteins/metabolism , Base Sequence , Gene Expression Regulation, Viral , Gene Knockdown Techniques , HEK293 Cells , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Humans , Inverted Repeat Sequences , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Small Interfering/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins/genetics , Transcription Factors , Tumor Necrosis Factor-alpha/physiology
10.
Cell Signal ; 26(5): 933-41, 2014 May.
Article in English | MEDLINE | ID: mdl-24447911

ABSTRACT

The omega-3 polyunsaturated fatty acids (ω-3 fatty acids) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to inhibit or delay the progression of cardiovascular diseases, including myocardial fibrosis. Recently we reported that angiotensin II (Ang II) promotes cardiac fibroblast (CF) migration by suppressing the MMP regulator reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), through a mechanism dependent on AT1, ERK, and Sp1. Here we investigated the role of miR-21 in Ang II-mediated RECK suppression, and determined whether the ω-3 fatty acids reverse these effects. Ang II induced miR-21 expression in primary mouse cardiac fibroblasts (CFs) via ERK-dependent AP-1 and STAT3 activation, and while a miR-21 inhibitor reversed Ang II-induced RECK suppression, a miR-21 mimic inhibited both RECK expression and Ang II-induced CF migration. Moreover, Ang II suppressed the pro-apoptotic PTEN, and the ERK negative regulator Sprouty homologue 1 (SPRY1), but induced the metalloendopeptidase MMP2, all in a manner that was miR-21-dependent. Further, forced expression of PTEN inhibited Akt phosphorylation, Sp1 activation, and MMP2 induction. Notably, while both EPA and DHA reversed Ang II-mediated RECK suppression, DHA appeared to be more effective, and reversed Ang II-induced miR-21 expression, RECK suppression, MMP2 induction, and CF migration. These results indicate that Ang II-induced CF migration is differentially regulated by miR-21-mediated MMP induction and RECK suppression, and that DHA has the potential to upregulate RECK, and therefore may exert potential beneficial effects in cardiac fibrosis.


Subject(s)
Angiotensin II/pharmacology , Docosahexaenoic Acids/pharmacology , Fibroblasts/drug effects , GPI-Linked Proteins/metabolism , 3' Untranslated Regions , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Movement/drug effects , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/genetics , Male , Matrix Metalloproteinase 2/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , MicroRNAs/metabolism , PTEN Phosphohydrolase/metabolism , Phosphoproteins/metabolism , Phosphorylation/drug effects , Promoter Regions, Genetic , STAT3 Transcription Factor/metabolism , Transcription Factor AP-1/metabolism
11.
Nat Commun ; 3: 1034, 2012.
Article in English | MEDLINE | ID: mdl-22948816

ABSTRACT

Mast cells are known effector cells in allergic and inflammatory diseases, but their precise roles in intestinal inflammation remain unknown. Here we show that activation of mast cells in intestinal inflammation is mediated by ATP-reactive P2X7 purinoceptors. We find an increase in the numbers of mast cells expressing P2X7 purinoceptors in the colons of mice with colitis and of patients with Crohn's disease. Treatment of mice with a P2X7 purinoceptor-specific antibody inhibits mast cell activation and subsequent intestinal inflammation. Similarly, intestinal inflammation is ameliorated in mast cell-deficient Kit(W-sh/W-sh) mice, and reconstitution with wild-type, but not P2x7(-/-) mast cells results in susceptibility to inflammation. ATP-P2X7 purinoceptor-mediated activation of mast cells not only induces inflammatory cytokines, but also chemokines and leukotrienes, to recruit neutrophils and subsequently exacerbate intestinal inflammation. These findings reveal the role of P2X7 purinoceptor-mediated mast cell activation in both the initiation and exacerbation of intestinal inflammation.


Subject(s)
Adenosine Triphosphate/immunology , Crohn Disease/immunology , Intestines/immunology , Mast Cells/immunology , Receptors, Purinergic P2X7/immunology , Animals , Cells, Cultured , Crohn Disease/genetics , Cytokines/genetics , Cytokines/immunology , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Purinergic P2X7/genetics
12.
Biochem J ; 447(3): 449-55, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-22849325

ABSTRACT

The mammalian transcriptional factors, Cdx1 and Cdx2 (Cdx is caudal-type homeobox) are paralogues and critical for the cellular differentiation of intestinal or colorectal epithelia. It has been reported previously that in Cdx1 transgenic or knockout mice, endogenous Cdx2 levels are inversely correlated with Cdx1 levels. Recently, we found that exogenous Cdx1 expression can suppress Cdx2 in a human colorectal tumour cell line, SW480, although the underlying molecular mechanisms were unclear. In the present study, we show that several microRNAs induced by exogenous Cdx1 expression directly bind to the CDX2 mRNA 3'UTR (untranslated region) to destabilize these transcripts, finally leading to their degradation. Using microarray analysis, we found that several miRNAs that were computationally predicted to target CDX2 mRNAs are up-regulated by exogenous Cdx1 expression in SW480 cells. Among these molecules, we identified miR-9, miR-16 and miR-22 as having the potential to suppress Cdx2 through the binding of the 3'UTR to its transcript. Importantly, simultaneous mutations of both the miR-9- and miR-16-binding sites in the CDX2 3'UTR were shown to be sufficient to block Cdx2 suppression. The results of the present study suggest a unique feature of miRNAs in which they contribute to homoeostasis by limiting the levels of transcription factors belonging to the same gene family.


Subject(s)
Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/metabolism , MicroRNAs/physiology , CDX2 Transcription Factor , Cell Line, Tumor , Colorectal Neoplasms , HEK293 Cells , Homeodomain Proteins/genetics , Humans , MicroRNAs/biosynthesis , MicroRNAs/genetics , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics
13.
J Biol Chem ; 287(15): 11924-33, 2012 Apr 06.
Article in English | MEDLINE | ID: mdl-22334708

ABSTRACT

We have previously shown that DPF2 (requiem/REQ) functions as a linker protein between the SWI/SNF complex and RelB/p52 NF-κB heterodimer and plays important roles in NF-κB transactivation via its noncanonical pathway. Using sensitive 293FT reporter cell clones that had integrated a SWI/SNF-dependent NF-κB reporter gene, we find in this study that the overexpression of DPF1, DPF2, DPF3a, DPF3b, and PHF10 significantly potentiates the transactivating activity of typical NF-κB dimers. Knockdown analysis using 293FT reporter cells that endogenously express these five proteins at low levels clearly showed that DPF3a and DPF3b, which are produced from the DPF3 gene by alternative splicing, are the most critical for the RelA/p50 NF-κB heterodimer transactivation induced by TNF-α stimulation. Our data further show that this transactivation requires the SWI/SNF complex. DPF3a and DPF3b are additionally shown to interact directly with RelA, p50, and several subunits of the SWI/SNF complex in vitro and to be co-immunoprecipitated with RelA/p50 and the SWI/SNF complex from the nuclear fractions of cells treated with TNF-α. In ChIP experiments, we further found that endogenous DPF3a/b and the SWI/SNF complex are continuously present on HIV-1 LTR, whereas the kinetics of RelA/p50 recruitment after TNF-α treatment correlate well with the viral transcriptional activation levels. Additionally, re-ChIP experiments showed DPF3a/b and the SWI/SNF complex associate with RelA on the endogenous IL-6 promoter after TNF-α treatment. In conclusion, our present data indicate that by linking RelA/p50 to the SWI/SNF complex, DPF3a/b induces the transactivation of NF-κB target gene promoters in relatively inactive chromatin contexts.


Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation , NF-kappa B p50 Subunit/metabolism , Transcription Factor RelA/metabolism , Transcription Factors/physiology , Transcriptional Activation , Binding Sites , Cell Line , Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation , DNA-Binding Proteins/metabolism , Genes, Reporter , Humans , Kinetics , Luciferases/biosynthesis , Luciferases/genetics , Multiprotein Complexes/metabolism , Protein Binding , Protein Isoforms/metabolism , Protein Isoforms/physiology , Protein Structure, Tertiary , Protein Transport , Response Elements , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/physiology
14.
Nucleic Acids Res ; 40(8): e58, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22259037

ABSTRACT

MicroRNAs (miRNAs) are involved in various biological processes and human diseases. The development of strong low-molecular weight inhibitors of specific miRNAs is thus expected to be useful in providing tools for basic research or in generating promising new therapeutic drugs. We have previously described the development of 'Tough Decoy (TuD) RNA' molecules, which achieve the long-term suppression of specific miRNA activity in mammalian cells when expressed from a lentivirus vector. In our current study, we describe new synthetic miRNA inhibitors, designated as S-TuD (Synthetic TuD), which are composed of two fully 2'-O-methylated RNA strands. Each of these strands includes a miRNA-binding site. Following the hybridization of paired strands, the resultant S-TuD forms a secondary structure with two stems, which resembles the corresponding TuD RNA molecule. By analyzing the effects of S-TuD against miR-21, miR-200c, miR-16 and miR-106b, we have elucidated the critical design features of S-TuD molecules that will provide optimum inhibitory effects following transfection into human cell lines. We further show that the inhibitory effects of a single transfection of S-TuD-miR200c are quite long-lasting (>7 days) and induce partial EMT, the full establishment of which requires 11 days when using a lentivirus vector that expresses TuD-miR200c continuously.


Subject(s)
MicroRNAs/antagonists & inhibitors , RNA/chemistry , Binding Sites , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Humans , MicroRNAs/chemistry , Nucleic Acid Conformation , Nucleic Acid Hybridization , RNA/metabolism , Transfection
15.
Mol Ther ; 19(6): 1107-15, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21386827

ABSTRACT

Vaccinia virus, once widely used for smallpox vaccine, has recently been engineered and used as an oncolytic virus for cancer virotherapy. Their replication has been restricted to tumors by disrupting viral genes and complementing them with products that are found specifically in tumor cells. Here, we show that microRNA (miRNA) regulation also enables tumor-specific viral replication by altering the expression of a targeted viral gene. Since the deletion of viral glycoprotein B5R not only decreases viral pathogenicity but also impairs the oncolytic activity of vaccinia virus, we used miRNA-based gene regulation to suppress B5R expression through let-7a, a miRNA that is downregulated in many tumors. The expression of B5R and the replication of miRNA-regulated vaccinia virus (MRVV) with target sequences complementary to let-7a in the 3'-untranslated region (UTR) of the B5R gene depended on the endogenous expression level of let-7a in the infected cells. Intratumoral administration of MRVV in mice with human cancer xenografts that expressed low levels of let-7a resulted in tumor-specific viral replication and significant tumor regression without side effects, which were observed in the control virus. These results demonstrate that miRNA-based gene regulation is a potentially novel and versatile platform for engineering vaccinia viruses for cancer virotherapy.


Subject(s)
Glycoproteins/metabolism , MicroRNAs/genetics , Oncolytic Viruses/physiology , Vaccinia virus/physiology , 3' Untranslated Regions/genetics , Animals , Caco-2 Cells , Cell Line, Tumor , Female , Glycoproteins/genetics , HeLa Cells , Humans , Mice , Mice, SCID , Oncolytic Viruses/genetics , Vaccinia virus/genetics , Virus Replication/genetics , Virus Replication/physiology , Xenograft Model Antitumor Assays
16.
Bioconjug Chem ; 22(1): 42-9, 2011 Jan 19.
Article in English | MEDLINE | ID: mdl-21141919

ABSTRACT

Small interfering RNA (siRNA) is a noncoding RNA with considerable potential as a new therapeutic drug for intractable diseases. siRNAs can be rationally designed and synthesized if the sequences of the disease-causing genes are known. In this paper, we describe the synthesis and properties of siRNAs modified with biaryl units. We found that incorporation of biaryl units into the 5' and 3' ends of sense and antisense strands of siRNA duplexes improved strand selectivity and nuclease resistance.


Subject(s)
RNA, Antisense/chemistry , RNA, Antisense/genetics , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Ribonucleases/metabolism , Animals , Base Sequence , Gene Silencing , Hepacivirus/physiology , Luciferases/genetics , RNA, Antisense/metabolism , RNA, Small Interfering/metabolism , Virus Replication/genetics
17.
Cancer Res ; 71(5): 1680-9, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21189327

ABSTRACT

The chromatin remodeling complex SWI/SNF is an important epigenetic regulator that includes one Brm or BRG1 molecule as catalytic subunit. Brm and BRG1 do not function identically, so this complex can regulate gene expression either positively or negatively, depending on the promoter to which it is recruited. Notably, Brm attenuation due to posttranscription suppression occurs often in human tumor cells, in which this event contributes to their oncogenic potential. Here, we report that the 3'-untranslated region of Brm mRNA has two sites that are efficiently targeted by the microRNAs miR-199a-5p and -3p, revealing a novel mechanism for modulation of Brm-type SWI/SNF activity. Computational mapping of the putative promoter region of miR-199a-2 (miPPR-199a-2) has defined it as the major contributing genetic locus for miR-199a-5p and-3p production in these tumor cell lines. We validated this predicted region by direct promoter analysis to confirm that Egr1 is a strong positive regulator of the miR-199a-2 gene. Importantly, we also showed that Egr1, miR-199a-5p, and miR-199a-3p are expressed at high levels in Brm-deficient tumor cell lines but only marginally in Brm-expressing tumor cells. Finally, we also obtained evidence that Brm negatively regulates Egr1. Together, our results reveal that miR-199a and Brm form a double-negative feedback loop through Egr1, leading to the generation of these two distinct cell types during carcinogenesis. This mechanism may offer a partial explanation for why miR-199a-5p and -3p have been reported to be either upregulated or downregulated in a variety of tumors.


Subject(s)
Early Growth Response Protein 1/genetics , Feedback, Physiological/physiology , Gene Expression Regulation, Neoplastic/genetics , MicroRNAs/genetics , Neoplasms/genetics , Transcription Factors/genetics , 3' Untranslated Regions , Base Sequence , Blotting, Western , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Humans , Molecular Sequence Data , Neoplasms/metabolism , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection
18.
Bioorg Med Chem Lett ; 20(24): 7299-302, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-21067927

ABSTRACT

MicroRNAs (miRNAs) are single-stranded non-coding RNAs composed of 20-23 nucleotides. They are initially transcribed in the nucleus as pri-miRNAs. After processing, one strand from the miRNA duplex (miR-5p/miR-3p duplex) is loaded onto the RNA-induced silencing complex (RISC) to produce a functional, mature miRNA that inhibits the expression of multiple target genes. In the case of some miRNAs, both strands can be equally incorporated into the RISC as single strands, and both strands can function as mature miRNAs. Thus, a technique for selective expression of miR-5p and miR-3p strands is required to identify distinct targets of miRNAs. In this Letter, we report the synthesis and properties of miRNA duplexes carrying biaryl units at the 5'-terminus of one strand. We found that incorporation of biaryl units at the 5'-terminus of one strand of miRNA duplexes induced strand specificity in these duplexes. Further, we succeeded in identifying endogenous mRNA targets for each strand of the duplex by using the biaryl-modified miRNA duplexes.


Subject(s)
MicroRNAs/metabolism , Naphthalenes/chemistry , Base Sequence , MicroRNAs/chemistry , MicroRNAs/genetics , RNA Interference , RNA-Induced Silencing Complex/chemistry , RNA-Induced Silencing Complex/metabolism
19.
J Biol Chem ; 285(29): 21951-60, 2010 Jul 16.
Article in English | MEDLINE | ID: mdl-20460684

ABSTRACT

The SWI/SNF chromatin remodeling complex plays pivotal roles in mammalian transcriptional regulation. In this study, we identify the human requiem protein (REQ/DPF2) as an adaptor molecule that links the NF-kappaB and SWI/SNF chromatin remodeling factor. Through in vitro binding experiments, REQ was found to bind to several SWI/SNF complex subunits and also to the p52 NF-kappaB subunit through its nuclear localization signal containing the N-terminal region. REQ, together with Brm, a catalytic subunit of the SWI/SNF complex, enhances the NF-kappaB-dependent transcriptional activation that principally involves the RelB/p52 dimer. Both REQ and Brm were further found to be required for the induction of the endogenous BLC (CXCL13) gene in response to lymphotoxin stimulation, an inducer of the noncanonical NF-kappaB pathway. Upon lymphotoxin treatment, REQ and Brm form a larger complex with RelB/p52 and are recruited to the BLC promoter in a ligand-dependent manner. Moreover, a REQ knockdown efficiently suppresses anchorage-independent growth in several cell lines in which the noncanonical NF-kappaB pathway was constitutively activated. From these results, we conclude that REQ functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in noncanonical NF-kappaB transcriptional activation and its associated oncogenic activity.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Signal Transduction , Transcription Factor RelB/metabolism , Transcription Factors/metabolism , Cell Line , Chemokine CXCL13/genetics , Chemokine CXCL13/metabolism , DNA Helicases/metabolism , DNA-Binding Proteins/chemistry , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Lymphotoxin-alpha/pharmacology , Neoplasms/metabolism , Neoplasms/pathology , Nuclear Proteins/metabolism , Protein Binding/drug effects , Protein Subunits/metabolism , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Transcriptional Activation/genetics
20.
J Virol ; 83(22): 11569-80, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19726504

ABSTRACT

To elucidate the epigenetic regulation of Tat-independent human immunodeficiency virus (HIV) transcription following proviral integration, we constructed an HIV type 1 (HIV-1)-based replication-defective viral vector that expresses a reporter green fluorescent protein (GFP) product from its intact long terminal repeat (LTR). We transduced this construct into human tumor cell lines that were either deficient in or competent for the Brm-type SWI/SNF complex. One day after transduction, single cells that expressed GFP were sorted, and the GFP expression profiles originating from each of these clones were analyzed. Unlike clones of the SWI/SNF-competent cell line, which exhibited clear unimodal expression patterns in all cases, many clones originating from Brm-deficient cell lines either showed a broad-range distribution of GFP expression or were fully silenced. The resorting of GFP-negative populations of these isolated clones showed that GFP silencing is either reversible or irreversible depending upon the proviral integration sites. We further observed that even in these silenced clones, proviral gene transcription initiates to accumulate short transcripts of around 60 bases in length, but no elongation occurs. We found that this termination is caused by tightly closed nucleosome-1 (nuc-1) at the 5' LTR. Also, nuc-1 is remodeled by exogenous Brm in some integrants. From these results, we propose that Brm is required for the occasional transcriptional elongation of the HIV-1 provirus in the absence of Tat. Since the Brm-type SWI/SNF complex is expressed at marginal levels in resting CD4+ T cells and is drastically induced upon CD4+ T-cell activation, we speculate that it plays crucial roles in the early Tat-independent phase of HIV transcription in affected patients.


Subject(s)
Chromatin Assembly and Disassembly/genetics , HIV-1/genetics , Transcriptional Elongation Factors/genetics , tat Gene Products, Human Immunodeficiency Virus/genetics , Cell Line, Tumor , Genetic Vectors/genetics , HIV-1/physiology , Humans , RNA, Viral/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/physiology , Transcription, Genetic/genetics , Transcriptional Elongation Factors/physiology , Transduction, Genetic , Virus Replication/genetics , tat Gene Products, Human Immunodeficiency Virus/physiology
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