Takeshi Haraguchi

Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells

Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

Purpose: To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH). Experimental Design: Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21–expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps. Results: In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 in vivo. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation. Conclusion: Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.

Extracellular ATP mediates mast cell-dependent intestinal inflammation through P2X7 purinoceptors

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 Crohns 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 KitW-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.

Frequent Loss of Brm Expression in Gastric Cancer Correlates with Histologic Features and Differentiation State

The mammalian SWI/SNF chromatin remodeling complex, an essential epigenetic regulator, contains either a single Brm or BRG1 molecule as its catalytic subunit. We observed frequent loss of Brm expression but not of BRG1 in human gastric cancer cell lines. Treatment with histone deacetylase inhibitor rescued Brm expression, indicating epigenetic regulation of this gene, and an RNA interference-based colony formation assay revealed antioncogenic properties of Brm. Brm immunostaining of 89 primary gastric cancers showed an obvious reduction in 60 cases (67%) and a severe decrease in 37 cases (42%). Loss of Brm is frequent in the major gastric cancer types (well- or moderately-differentiated tubular adenocarcinoma and poorly-differentiated adenocarcinoma) and positively correlates with the undifferentiated state. Among the minor gastric cancer types, Brm expression persists in signet-ring cell carcinoma and mucinous adenocarcinoma, but a marked decrease is observed in papillary adenocarcinoma. Intestinal metaplasia never shows decreased expression, indicating that Brm is a valid marker of gastric oncogenesis. In contrast, BRG1 is retained in most cases; a concomitant loss of BRG1 and Brm is rare in gastric cancer, contrary to other malignancies. We further show that Brm is required for villin expression, a definitive marker of intestinal metaplasia and differentiation. Via regulating such genes important for gut differentiation, Brm should play significant roles in determining the histologic features of gastric malignancy.

SWI/SNF complex is essential for NRSF-mediated suppression of neuronal genes in human nonsmall cell lung carcinoma cell lines

Mammalian chromatin remodeling factor, SWI/SNF complex contains a single molecule of either Brm or BRG1 as the ATPase catalytic subunit. Here, we show that the SWI/SNF complex forms a larger complex with neuron-restrictive silencer factor (NRSF) and its corepressors, mSin3A and CoREST, in human nonsmall cell lung carcinoma cell lines. We also demonstrate that the strong transcriptional suppression of such neuron-specific genes as synaptophysin and SCG10 by NRSF in these non-neural cells requires the functional SWI/SNF complex; these neuronal genes were elevated in cell lines deficient in both Brm and BRG1, whereas retrovirus vectors expressing siRNAs targeting integral components of SWI/SNF complex (Brm/BRG1 or Ini1) induced expression of these neuronal genes in SWI/SNF-competent cell lines. In cell lines deficient in both Brm and BRG1, exogenous Brm or BRG1 suppressed expression of these neuronal genes in an ATP-dependent manner and induced efficient and specific deacetylation of histone H4 around the NRSF binding site present in the synaptophysin gene by a large complex containing the recruited functional SWI/SNF complex. Patients with Brm/BRG1-deficient lung carcinoma have been reported to carry poor prognosis; derepression of NRSF-regulated genes including these neuron-specific genes could contribute to enhance tumorigenicity and also would provide selective markers for Brm/BRG1-deficient tumors.

The metabolism of biphenyl structures in lignin by the soil bacterium (Pseudomonas paucimobilis SYK-6)

In the soil bacterium (Pseudomonas paucimobilis SYK‐6), the metabolism of DDVA (biphenyl structure of lignin) and syringic acid (characteristic aromatic ring in hardwood lignin) proceeds via a common intermediate, 3‐methylgallic acid. Protocatechuate is also an intermediate in the metabolism of vanillate and p‐hydroxybenzoic acid. 3‐Methylgallic acid and protocatechuate are the final aromatic intermediates in lignin microbial degradation and these compounds are substrates of protocatechuate‐4,5‐dioxygenase, which is a key enzyme in obtaining metabolic energy from various structures of lignin in this bacterium.

A potent 2'-O-methylated RNA-based microRNA inhibitor with unique secondary structures.

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.

Requiem Protein Links RelB/p52 and the Brm-type SWI/SNF Complex in a Noncanonical NF-κB Pathway

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-κB 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-κB 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-κB-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-κB 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-κB 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-κB transcriptional activation and its associated oncogenic activity.

Cdx2 and the Brm-type SWI/SNF complex cooperatively regulate villin expression in gastrointestinal cells

In our recent study showing a correlation between Brm-deficiency and undifferentiated status of gastric cancer, we found that the Brm-type SWI/SNF complex is required for villin expression. To elucidate intestinal villin regulation more precisely, we here analyzed structure and function of the promoter of human villin. About 1.1 kb upstream of the determined major transcription start site, we identified a highly conserved region (HCR-Cdx) among mammals, which contains two binding sites for Cdx. Expression analyses of 30 human gastrointestinal cell lines suggested that villin is regulated by Cdx2. Introduction of Cdx family genes into colorectal SW480 cells revealed that villin is strongly induced strongly by Cdx2, moderately by Cdx1, and marginally by Cdx4. Knockdown of Cdx2 in SW480 cells caused a clear downregulation of villin, and reporter assays showed that HCR-Cdx is crucial for Cdx2-dependent and Brm-dependent villin expression. Immunohistochemical analyses of gastric intestinal metaplasia and cancer revealed that villin and Cdx2 expression are tightly coupled. GST pull-down assays demonstrated a direct interaction between Cdx2 and several SWI/SNF subunits. Chromatin immunoprecipitation analyses showed the recruitment of Cdx2 and Brm around HCR-Cdx. From these results, we concluded that Cdx2 regulates intestinal villin expression through recruiting Brm-type SWI/SNF complex to the villin promoter.

Brm transactivates the telomerase reverse transcriptase (TERT) gene and modulates the splicing patterns of its transcripts in concert with p54nrb

We report that a DBHS (Drosophila behaviour, human splicing) family protein, p54(nrb), binds both BRG1 (Brahma-related gene 1) and Brm (Brahma), catalytic subunits of the SWI/SNF (switch/sucrose non-fermentable) chromatin remodelling complex, and also another core subunit of this complex, BAF60a. The N-terminal region of p54(nrb) is sufficient to pull-down other core subunits of the SWI/SNF complex, suggesting that p54(nrb) binds SWI/SNF-like complexes. PSF (polypyrimidine tract-binding protein-associated splicing factor), another DBHS family protein known to directly bind p54(nrb), was also found to associate with the SWI/SNF-like complex. When sh (short hairpin) RNAs targeting Brm were retrovirally expressed in a BRG1-deficient human cell line (NCI-H1299), the resulting clones showed down-regulation of the TERT (telomerase reverse transcriptase) gene and an enhancement of ratios of exon-7-and-8-excluded TERT mRNA that encodes a beta-site-deleted inactive protein. All of these clones display growth arrest within 2 months of the Brm-knockdown. In NCI-H1299 cells, Brm, p54(nrb), PSF and RNA polymerase II phosphorylated on CTD (C-terminal domain) Ser(2) specifically co-localize at a region incorporating an alternative splicing acceptor site of TERT exon 7. These findings suggest that, at the TERT gene locus in human tumour cells containing a functional SWI/SNF complex, Brm, and possibly BRG1, in concert with p54(nrb), would initiate efficient transcription and could be involved in the subsequent splicing of TERT transcripts by accelerating exon-inclusion, which partly contributes to the maintenance of active telomerase.