Regulate Target Gene Expression Research Articles

Oct1 Is a Switchable, Bipotential Stabilizer of Repressed and Inducible Transcriptional States

Little is known regarding how the Oct1 transcription factor regulates target gene expression. Using murine fibroblasts and two target genes, Polr2a and Ahcy, we show that Oct1 recruits the Jmjd1a/KDM3A lysine demethylase to catalyze the removal of the inhibitory histone H3K9 dimethyl mark and block repression. Using purified murine T cells and the Il2 target locus, and a colon cancer cell line and the Cdx2 target locus, we show that Oct1 recruits the NuRD chromatin-remodeling complex to promote a repressed state, but in a regulated manner can switch to a different capacity and mediate Jmjd1a recruitment to block repression. These findings indicate that Oct1 maintains repression through a mechanism involving NuRD and maintains poised gene expression states through an antirepression mechanism involving Jmjd1a. We propose that, rather than acting as a primary trigger of gene activation or repression, Oct1 is a switchable stabilizer of repressed and inducible states.

MicroRNA expression profiles in human colorectal cancers with liver metastases

At present, a full understanding of the mechanisms by which colorectal cancer (CRC) distant metastases form is still beyond our reach because of the intricate regulation of gene expression. MicroRNAs (miRNAs) are shown to be involved in various human diseases including cancers through negative regulation of target gene expression at the post-transcriptional level. However, there are only a few studies on the roles of miRNA aberrations in liver metastasis of human colorectal cancer. To identify miRNA expression patterns associated with liver metastasis in human colorectal cancer, the miRNA expression profiles of colorectal cancer tissues with liver metastasis and their non-metastatic counterparts were studied using microRNA microarrays and further confirmed by quantitative RT-PCR. We show that 28 miRNAs are differentially expressed in the colorectal carcinomas with liver metastasis compared to the non-metastatic counterparts. Of these, 4 miRNAs including miR-150*, miR-125b-2*, miR-1179 and miR-139-3p were up-regulated in colorectal cancers with liver metastasis while the others were down-regulated. The target genes of selected deregulated miRNAs were predicted through bioinformatic techniques with two functional analyses, gene ontology and KEGG analysis, which showed that categories of high enrichment GOs and specific pathways targeted by dysregulated miRNAs were involved in liver metastasis during human colorectum carcinogenesis. Our results indicated that miRNAs are not only involved in carcinogenesis of colorectum, but may also participate in the progression such as with liver metastases in human colorectal cancers.

Abstract P3-04-01: Integrated Analysis of Epigenetic Signatures Associated with Inflammatory Breast Cancer

Abstract Inflammatory breast cancer (IBC) is the most aggressive manifestation of primary epithelial breast cancer and differs from non-IBC both from a clinical and a biological perspective. The high metastatic potential of IBC is highlighted by the low 5-year survival rate of 50%. The biological features that make IBC unique are its rapid local growth, its high angioinvasive capability with early metastasis and its aggressive behavior from inception. These differences could be the result of key changes in the epigenetic profile of IBC. To address this issue, we compared the levels of epigenetic regulation in IBC (N=19) and non-IBC (N=43) samples at the methylome (DNA methylation), miRome (miRNA) and transcriptome (mRNA) level using high-throughput molecular technologies. Differences in gene expression profiles allowed the classification of samples according to tumor subtype (IBC vs non-IBC). The analysis of 27,578 CpG loci demonstrated that the largest variation in methylation levels between samples was attributable to differences in metastatic status (P=0.026) and patients’ age (P=0.049). About 2% of all interrogated CpG loci was differentially methylated in IBC and non-IBC (FDR <0.1). Annotated gene functions for these CpG loci were enriched for biological processes related to inflammation and immune response. Using a whole genome approach, we observed significant, negative correlations between methylation and mRNA levels for 38% of genes. For 8% of genes, a significant, positive correlation was observed. Interrogated CpG loci for these genes were more frequently located in the gene body (>500 bp from TSS) (P=0.008). Our analysis of 322 miRNAs showed that the largest variation in miRNA levels between samples was attributable to differences in ER status (P=0.001) and histological grade (P=0.002). About 4% of all miRNAs was expressed at different levels in IBC and non-IBC. Using a whole genome approach, we identified 17,295 of highly correlated miRNA-mRNA pairs for these IBC-associated miRNAs. Based on this miRNA target gene information, we were able to detect a variety of biological processes that may indicate function of these miRNAs and to indicate potential associations of these miRNAs with prognosis in breast cancer. Furthermore, the comparison of mRNA levels of key miRNA processing genes revealed the upregulation of Ago2 (P=0.002) and the downregulation of Dicer (P=0.004) in IBC when compared to non-IBC, which may suggest higher RISC activities and, therefore, that miRNAs regulate target gene expression in IBC with higher efficiency. In conclusion, this study represents the first integrated analysis of epigenetic signatures in IBC. Using high-throughput technologies, we were able to identify a number of epigenetic markers specifically associated with IBC. A global initiative has been taken to set up an IBC tissue repository that will allow the confirmation of these kinds of studies on a larger IBC sample population. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-01.

The Suppression of CRMP2 Expression by Bone Morphogenetic Protein (BMP)-SMAD Gradient Signaling Controls Multiple Stages of Neuronal Development

The formation of the functional mammalian cerebral cortex requires a concerted control of neurogenesis, neuronal migration, and neuronal morphogenesis. However, molecular mechanisms that control these processes are not well understood. We have found that the BMP signaling downstream transcription factor SMAD1 and CRMP2 (collapsin response mediator protein-2) are inversely and complementarily expressed in the developing neocortex. BMPs can suppress CRMP2 expression in cortical cells. Our ChIP assay demonstrates that both SMAD1 and -4 bind to CRMP2 promoter in the neocortex, and overexpression of SMAD1 and 4 in vivo suppresses CRMP2 expression. RNA interference of CRMP2 and overexpression of dominant negative forms of CRMP2 in utero cause accumulation of multipolar cells in the ventricular, subventricular, and intermediate zones and suppresses neurite outgrowth, suggesting that CRMP2 is required for multipolar to bipolar transition for directional neuronal migration and neurite outgrowth. Thus, our study reveals a novel mechanism that the BMP-SMAD signaling pathway controls neuronal migration and neurite outgrowth by suppressing the transcription of CRMP2.

Isoflavones suppress cyclic adenosine 3′,5′-monophosphate regulatory element-mediated transcription in osteoblastic cell line

Soy isoflavones have been implicated to exert benefit on bone loss in postmenopausal women. Isoflavones can induce estrogen response element-mediated transcription in osteoblastic cells. In the present study, we investigate whether isoflavones genistein and daidzein regulate target gene transcription through cAMP regulatory element (CRE) in osteoblastic cells. It was found that 17β-estradiol (E 2), genistein and daidzein suppressed the transcriptional activity of CRE-luciferase reporter gene in human osteoblastic cell line MG-63 cells. E 2 and genistein but not daidzein inhibited the cAMP analogue 8-Br cAMP-induced transcription of CRE reporter gene. Both genistein and E 2 inhibited basal and cAMP-induced mRNA levels of endogenous estrogen responsive genes containing CRE/CRE-like elements in their promoter regions, including interleukin (IL) 8 and serum- and glucocorticoid-inducible kinase 1 (SGK1). Daidzein inhibited basal and cAMP-induced IL-8, but not SGK1 mRNA expression. The inhibitory effects of E 2, genistein and daidzein on CRE-mediated transcription activity were enhanced by estrogen receptor (ER) α overexpression in MG-63 cells, which could be blocked by nonselective ER antagonists ICI182780, 4-OH tamoxifen and specific ERα antagonist MPP. Genistein and daidzein, but not E 2 treatment, caused a significant decrease in CRE-mediated transcription activity in ERβ-transfected MG-63 cells, which could be blocked by ICI182780, 4-OH tamoxifen and the selective ERβ antagonist ( R, R)-5,11-diethyl-5.6,11,12-tetradro-2,8-chrysenediol. Our results indicate that isoflavones genistein and daidzein might modulate bone remodeling through ERs by regulating target gene expression through the CRE motifs.

Synergistic apoptosis induction in leukemic cells by miR-15a/16-1 and arsenic trioxide

MicroRNAs (miRNAs) are small noncoding RNAs that regulate target gene expression through translation repression or messenger RNA degradation. MiR-15a and 16-1 form a cluster at the chromosomal region 13q14, which is frequently deleted or down-regulated in chronic lymphocytic leukemia. Arsenic trioxide (As2O3, ATO) has been successfully applied to treat acute promyelocytic leukemia (APL). Its combination with other drugs presented therapeutic activities in hematologic and solid tumors. Here we investigated the potential synergy between miR-15a/16-1 and ATO on Bcr-Abl positive leukemic K562 cells. In this study, we found that combination of miR-15a/16-1 and ATO synergistically induced growth inhibition and apoptosis in K562 cells. The apoptosis, at least in part, through regulating mitochondrial function including the release of cytochrome c and loss of mitochondrial transmembrane potential, also activation of caspase-3 and degradation of poly-adenosine diphosphate-ribose polymerase. However, the expression of Bcr-Abl was not affected by ATO and/or miR-15a/16-1. Moreover, apoptotic synergy between miR-15a/16-1 and ATO was observed in Bcr-Abl negative leukemic cell lines and primary leukemic cells. Taken together, these findings suggested that the combined regiment of miR-15a/16-1 and ATO might be a potential therapeutic remedy for the treatment of leukemia.

P13. Wnt signaling pathways regulate epithelial polarity and morphogenesis

Wnt proteins are highly conserved secreted proteins that are essential for development in all metazoan organisms. Wnt proteins bind to cell surface receptors and activates the β-catenin-dependent and -independent pathways. The β-catenin-dependent pathway regulates cell proliferation and differentiation through the regulation of target gene expression. The β-catenin-independent pathway is involved in cell migration and polarity. Knockout mouse studies revealed that both the β-catenin-dependent and -independent pathways of Wnt signaling are important in the epithelial morphogenesis of tubular organs such as the lungs, kidneys, guts, and mammary glands, but its molecular mechanism is poorly understood. Epithelial cells established apical–basal polarity and formed cyst with central lumen in three-dimensional culture. Epidermal growth factor (EGF) increased the size of cyst through the promotion of cell proliferation. Although Wnt3a, which is a representative ligand that activates the β-catenin-dependent pathway, did not affect cyst morphogenesis, combination of Wnt3a and EGF induced tube-like branching morphogenesis. Treatment with EGF and blebbistatin, a Myosin II inhibitor, induced branching morphogenesis similar to that induced by EGF and Wnt3a. Blebbistatin inhibited phosphorylation of paxillin, which is a component of focal adhesions. Knockdown of β-catenin or LRP6, which is a receptor of Wnt3a, inhibited branching morphogenesis induced by Wnt3a. Wnt5a, which is a representative ligand that activates the β-catenin-independent pathway, did not affect cyst morphology in the presence or absence of EGF. Knockdown of Wnt5a inhibited the formation of apical membrane domain of epithelial cells. ECM signaling from the basal cell surface is important for the decision of apical–basal polarity. Wnt5a was observed at the basal cell surface and Ror2, a Wnt5a receptor specifically localized basolateral cell surface in epithelial cyst. These results indicate that both the β-catenin-dependent and -independent pathways of Wnt signaling are involved in the regulation of epithelial polarity and morphogenesis through the distinct mechanisms.

Exon Junction Complex Subunits Are Required to Splice Drosophila MAP Kinase, a Large Heterochromatic Gene

The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns.

E0051 miR214 is upregulated during ventricular remodelling post MI

ObjectiveMicroRNAs (miRNAs) are endogeneous, single-stranded non-coding RNA molecules about 22 nucleotides long, del regulating target gene expression post-transcriptionally by base pairing with specific binding sites located in the 3' -...

P38 Regulates Expression of Osteoblast-specific Genes by Phosphorylation of Osterix

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

MicroRNA-155 regulates angiotensin II type 1 receptor expression and phenotypic differentiation in vascular adventitial fibroblasts

MicroRNAs (miRNAs), which are genomically encoded small RNAs, negatively regulate target gene expression at the post-transcriptional level. Our recent study indicated that microRNA-155 (miR-155) might be negatively correlated with blood pressure, and it has been suggested that miR-155-mediated target genes could be involved in the cardiovascular diseases. Bioinformatic analyses predict that angiotensin II type 1 receptor (AT 1R) is a miR-155 target gene. The present study investigated the potential role of miR-155 in regulating AT 1R expression and phenotypic differentiation in rat aortic adventitial fibroblasts (AFs). Luciferase assay demonstrated that miR-155 suppressed AT 1R 3′-UTR reporter construct activity. miR-155 overexpression in AFs did not reduce target mRNA levels, but significantly reduced target protein expression. In addition, AFs transfected with pSUPER/miR-155 exhibited reduced Ang II-induced ERK1/2 activation. miR-155 overexpression in cells attenuated Ang II-induced α-smooth muscle actin (α-SMA, produces myofibroblast) expression, but did not transform growth factor beta-1 (TGF-β1). This study demonstrated that miR-155 could have an important role in regulating adventitial fibroblast differentiation and contribute to suppression of AT 1R expression.

The Pancreatic and Duodenal Homeobox Protein PDX-1 Regulates the Ductal Specific Keratin 19 through the Degradation of MEIS1 and DNA Binding

BackgroundPancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE) homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19). In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation.Methodology/Principal FindingsHere, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH2-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome.Conclusions/SignificanceTaken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non-redundant mechanisms.

Heterodimers of NF-κB transcription factors DIF and Relish regulate antimicrobial peptide genes in Drosophila

The innate immune response in Drosophila involves the inducible expression of antimicrobial peptide genes mediated by the Toll and IMD signaling pathways. Dorsal and DIF act downstream of Toll, whereas Relish acts downstream of IMD to regulate target gene expression. Dorsal, DIF, and Relish are NF-kappaB-related transcription factors and function as obligate dimers, but it is not clear how the various dimer combinations contribute to the innate immune response. We systematically examined the dimerization tendency of these proteins through the use of transgenic assays. The results show that all combinations of homo- and heterodimers are formed, but with varying degrees of efficiency. The formation of the DIF-Relish heterodimer is particularly interesting because it may mediate signaling for the seemingly independent Toll and IMD pathways. By incorporating a flexible peptide linker, we specifically tested the functions of the DIF;Relish (a ; sign represents the peptide linker) linked heterodimer. Our results demonstrate that the linked heterodimer can activate target genes of both the Toll and IMD pathways. The DIF and Relish complex is detectable in whole animal extracts, suggesting that this heterodimer may function in vivo to increase the spectrum and level of antimicrobial peptide production in response to different infections.

MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.

HPeak: an HMM-based algorithm for defining read-enriched regions in ChIP-Seq data

BackgroundProtein-DNA interaction constitutes a basic mechanism for the genetic regulation of target gene expression. Deciphering this mechanism has been a daunting task due to the difficulty in characterizing protein-bound DNA on a large scale. A powerful technique has recently emerged that couples chromatin immunoprecipitation (ChIP) with next-generation sequencing, (ChIP-Seq). This technique provides a direct survey of the cistrom of transcription factors and other chromatin-associated proteins. In order to realize the full potential of this technique, increasingly sophisticated statistical algorithms have been developed to analyze the massive amount of data generated by this method.ResultsHere we introduce HPeak, a Hidden Markov model (HMM)-based Peak-finding algorithm for analyzing ChIP-Seq data to identify protein-interacting genomic regions. In contrast to the majority of available ChIP-Seq analysis software packages, HPeak is a model-based approach allowing for rigorous statistical inference. This approach enables HPeak to accurately infer genomic regions enriched with sequence reads by assuming realistic probability distributions, in conjunction with a novel weighting scheme on the sequencing read coverage.ConclusionsUsing biologically relevant data collections, we found that HPeak showed a higher prevalence of the expected transcription factor binding motifs in ChIP-enriched sequences relative to the control sequences when compared to other currently available ChIP-Seq analysis approaches. Additionally, in comparison to the ChIP-chip assay, ChIP-Seq provides higher resolution along with improved sensitivity and specificity of binding site detection. Additional file and the HPeak program are freely available at http://www.sph.umich.edu/csg/qin/HPeak.

Secreted Monocytic miR-150 Enhances Targeted Endothelial Cell Migration

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate target gene expression at the posttranscriptional level. Here, we report that secreted miRNAs can serve as signaling molecules mediating intercellular communication. In human blood cells and cultured THP-1 cells, miR-150 was selectively packaged into microvesicles (MVs) and actively secreted. THP-1-derived MVs can enter and deliver miR-150 into human HMEC-1 cells, and elevated exogenous miR-150 effectively reduced c-Myb expression and enhanced cell migration in HMEC-1 cells. In vivo studies confirmed that intravenous injection of THP-1 MVs significantly increased the level of miR-150 in mouse blood vessels. MVs isolated from the plasma of patients with atherosclerosis contained higher levels of miR-150, and they more effectively promoted HMEC-1 cell migration than MVs from healthy donors. These results demonstrate that cells can secrete miRNAs and deliver them into recipient cells where the exogenous miRNAs can regulate target gene expression and recipient cell function.

Downregulation of Dicer enhances tumor cell proliferation and invasion

miRNAs are non-coding, single-stranded RNAs that regulate target gene expression by repressing translation or promoting RNA cleavage. Dicer is an essential component of the miRNA processing machinery. To identify a role for miRNAs in tumorigenesis, we designed an adenovirus expressing small hairpin RNA (shRNA) to silence Dicer and globally suppress the maturation of miRNAs. We identified that the impairment of miRNA processing conferred an enhanced proliferative activity and invasive ability on each of three tumor cell lines in vitro. Inhibition of Dicer was associated with activation of p-Akt and enhanced expression of the cell cycle associating molecules, cyclin A and PCNA, as well as MMP-2 and MMP-9, proteins involved in tumor cell invasion. Adenoviral gene silencing of Dicer in subcutaneous MCF-7 xenografts significantly increased tumor growth in vivo compared to tumors infected with non-loading adenovirus. Increased tumor growth was associated with p-Akt activation and upregulation of cyclin A, PCNA MMP-2 and MMP-9. These findings demonstrate that global reduction of miRNA processing by silencing Dicer enhances tumor proliferation and invasion, and the p-Akt pathway may contribute to this phenotype via the downstream molecules, cyclin A, PCNA, MMP-2 and MMP-9.

The Poly(C)-Binding Protein-1 Regulates Expression of the Androgen Receptor

The androgen receptor (AR) is a ligand-dependent transcription factor, expressed in male and female reproductive organs, and essential for normal reproduction in both sexes. The levels of AR are tightly controlled in androgen-responsive cells in which it plays a central role in the regulation of target gene expression. The AR is abundantly expressed in human endometrial stromal cells (HESCs), but levels decline markedly after differentiation into decidual cells in vivo and in primary cultures. Decidualization profoundly down-regulated AR protein levels with no discernible effect on either AR mRNA or protein stability, suggesting that loss of the receptor was a consequence of translational inhibition. Here we show that HESCs express three RNA-binding proteins, Hu antigen R and the poly(C)-binding proteins PCBP1 and PCBP2, that reportedly target the 3'-untranslated region of AR transcripts. Only PCBP1 expression was enhanced in secretory endometrium in vivo and in decidualizing HESCs. Furthermore, knockdown of PCBP1 in decidualizing cells was sufficient to restore AR protein levels, indicating that loss of the AR protein is primarily the consequence of a translational block. PCBP1 also blocked AR translation in a cell-free system, although this did not require binding to the 3'-untranslated region of the receptor mRNA. Furthermore, knockdown of PCBP1 in the prostate cancer LNCaP cell line also increased AR protein. Therefore, PCBP1 plays a major role in the dynamic expression of AR in both male and female androgen-responsive cells.

Dicer is Required for the Normal Development of Sea Urchin,Hemicentrotus pulcherrimus

MicroRNAs are single-stranded RNA molecules with a length of 19-25 nucleotides, which play roles in various biological phenomena, including development, differentiation, apoptosis, by regulating target gene expression. Although the presence of microRNA molecules in sea urchin and the expression of genes involved in microRNA biogenesis during sea urchin development have been reported recently, the function of microRNA in sea urchin development remains to be elucidated. In this study, to understand the function of microRNA in the early development of sea urchin, we focused on Dicer, an essential enzyme for biosynthesis of mature microRNA. We determined the nucleotide sequence of cDNA for a Dicer homolog in the sea urchin, Hemicentrotus pulcherrimus, HpDcr, and found that functional domains of Dicer proteins are conserved in HpDcr. Analyses of its pattern of expression showed that HpDcr mRNA is expressed in embryos at all developmental stages analyzed, and seems to distribute asymmetrically at the morula and later stages. Knockdown of HpDcr resulted in anomalous morphogenesis, such as impairment of gastrulation and skeletogenesis at the mesenchyme blastula stage and later stages, and alteration of mRNA levels of cell type-specific genes. Thus, HpDcr plays important roles in morphogenesis in sea urchin embryos, suggesting that miRNA could be involved in the early development of sea urchin by regulating target gene expression.

Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.