Shinichiro Ohno

miR-92 is a key oncogenic component of the miR-17-92 cluster in colon cancer

MicroRNAs (miRNAs) belong to a class of endogenously expressed non‐coding small RNAs that function primarily as gene regulators. Growing evidence suggests that miRNAs play a significant role in tumor development, making them potential biomarkers for cancer diagnosis and prognosis. The miR‐17–92 cluster has emerged as an important locus, being highly overexpressed in several cancers in association with cancer development and progression. The miR‐17–92 miRNA cluster generates a single polycistronic primary transcript that yields six mature miRNAs: miR‐17, miR‐18a, miR‐19a, miR‐20a, miR‐19b, and miR‐92a. In colon cancer development, the pathophysiologic roles of these transcripts and their targets are largely unknown. In the present study, we performed copy number analyses of the six miRNAs transcribed from the miR‐17–92 cluster in colon tumor tissues. We determined that miR‐92a was transcribed at higher levels than the other five miRNAs in both adenomas and carcinoma. In addition, miR‐92a directly targeted the anti‐apoptotic molecule BCL‐2‐interacting mediator of cell death (BIM) in colon cancer tissues. An anti‐miR‐92a antagomir induced apoptosis of colon cancer‐derived cell lines. These data indicate that miR‐92a plays a pivotal role in the development of colorectal carcinoma. (Cancer Sci 2011; 102: 2264–2271)

Roles of exosomes and microvesicles in disease pathogenesis.

A variety of cells release membrane vesicles, such as exosomes and microvesicles (MVs), that are thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents throughout the body. There have been considerable efforts to use MVs as diagnostic or prognostic markers because their composition is reflective of minor physiological changes. Furthermore, recent studies demonstrate that MVs derived from infected cells and tumors contribute to disease pathogenesis. This review presents an overview of the potential roles of MVs with respect to clinical diagnosis and disease pathogenesis.

MicroRNA-18a modulates STAT3 activity through negative regulation of PIAS3 during gastric adenocarcinogenesis

Background:MicroRNA (miRNA, miR)-18a is a member of the miR-17–92 cluster, an important locus that is markedly overexpressed in several cancers and associated with cancer development and progression. However, the mechanism of action of the miR-17–92 cluster and its individual miRNAs are largely unknown.Methods and Results:In this study, we investigated the expression of the miR-17–92 cluster by in situ hybridisation (ISH) assay and copy-number analysis in gastric tissue microarray (TMA) specimens. We determined that miR-18a was present at higher levels than the other five miRNAs in the cluster. In addition, we identified Protein Inhibitor of Activated Signal Transducer and Activator of Transcription 3 (PIAS3) as a direct target of miR-18a in gastric cancer. miR-18a level was positively correlated with levels of Survivin, Bcl-xL, and c-Myc, which are downstream transcriptional targets of Signal Transducer and Activator of Transcription 3 (STAT3). STAT3-induced transcription can be negatively regulated by PIAS3; consistent with this, PIAS3 level was negatively correlated with levels of Survivin, Bcl-xL, and c-Myc.Conclusion:Our findings indicate that miR-18a acts as an oncogene and plays a role in gastric adenocarcinogenesis, at least in part by negatively regulating PIAS3 and thereby modulating expression of STAT3 target genes.

Focus on Extracellular Vesicles: Development of Extracellular Vesicle-Based Therapeutic Systems

Many types of cells release phospholipid membrane vesicles thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents. Extracellular vesicles (EVs) carry various proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs), like a “message in a bottle” to cells in remote locations. The encapsulated molecules are protected from multiple types of degradative enzymes in body fluids, making EVs ideal for delivering drugs. This review presents an overview of the potential roles of EVs as natural drugs and novel drug-delivery systems.

TLS-CHOP represses miR-486 expression, inducing upregulation of a metastasis regulator PAI-1 in human myxoid liposarcoma.

Myxoid liposarcomas (MLSs) are characterized by t(12;16)(q13;p11) translocation and expression of TLS-CHOP chimeric oncoprotein. However, the molecular functions of TLS-CHOP have not been fully understood. On the other hand, microRNAs (miRNAs) comprise an abundant class of endogenous small non-coding RNAs that negatively regulate the expression of their target genes, and are involved in many biological processes. It is now evident that dysregulation of miRNAs is an important step in the development of many cancers. To our knowledge, however, there have been no reports of the miRNAs involved in MLS tumorigenesis and development. In this study, we have found that miR-486 expression was repressed in TLS-CHOP-expressed NIH3T3 fibroblasts and MLS tissues, and exogenous overexpression of miR-486 repressed growth of MLS cells. Thus, downregulation of miR-486 may be an important process for MLS. In addition, we have identified plasminogen activator inhibitor-1 (PAI-1) as a novel target gene of miR-486. PAI-1 is a unique type of serine protease inhibitor and is known to be one of the key regulators of tumor invasion and metastasis. Furthermore, knockdown of PAI-1 by a specific small interfering RNA (siRNA) inhibited growth of MLS cells, suggesting that increased expression of PAI-1 by miR-486 repression is critical for survival of MLS cells. Collectively, these results suggest a novel essential molecular mechanism that TLS-CHOP activates PAI-1 expression by repression of miR-486 expression in MLS tumorigenesis and development.

Topical Use of Angiopoietin-like Protein 2 RNAi-loaded Lipid Nanoparticles Suppresses Corneal Neovascularization

Corneal neovascularization (CNV) is a sight-threatening condition that is encountered in various inflammatory settings including chemical injury. We recently confirmed that angiopoietin-like protein 2 (ANGPTL2) is a potent angiogenic and proinflammatory factor in the cornea, and we have produced a single-stranded proline-modified short hairpin anti-ANGPTL2 RNA interference molecule that is carried in a lipid nanoparticle (ANGPTL2 Li-pshRNA) for topical application. In this study, we have further examined the topical delivery and anti-ANGPTL2 activity of this molecule and have found that fluorescence-labeled ANGPTL2 Li-pshRNA eye drops can penetrate all layers of the cornea and that ANGPTL2 mRNA expression was dramatically inhibited in both epithelium and stroma at 12 and 24 hours after administration. We also examined the inhibitory effect of ANGPTL2 Li-pshRNA on CNV in a mouse chemical injury model and found that the area of angiogenesis was significantly decreased in corneas treated with ANGPTL2 Li-pshRNA eye drops compared to controls. Together, these findings indicate that this modified RNA interference agent is clinically viable in a topical formulation for use against CNV.

Exosome-Mediated Targeted Delivery of miRNAs

Many types of cells release phospholipid membrane vesicles that are thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents. These membrane vesicles, derived from the late endosomes, are called exosomes. Various proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs) are carried by exosomes to cells in remote locations, like a message in a bottle. Because they can protect encapsulated small RNAs from ribonucleases (RNases) in body fluid, exosomes represent ideal carriers for nucleic acid drugs. In addition, because exosomes are constructed from self components, they are predicted to have low antigenicity and toxicity, extremely important properties for carriers used in drug delivery. This article describes a protocol for using exosomes as carriers for RNA drug delivery systems.

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I-like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Correlation between number of renal cysts and aortic circumferences measured using autopsy material.

Although the presence of renal cysts has been reported to be associated with aortic aneurysm or dissection by imaging studies, an autopsy study has not been performed. Therefore, in our institute, recent consecutive adult autopsy cases (n=108, 64 males and 44 females) were reviewed. The circumferences and atherosclerosis ratios of both thoracic and abdominal aorta were individually measured and graded. The number of renal cysts was scored and graded. Age of subjects along with histories of smoking, hypertension, and diabetes mellitus were confirmed. Multiple linear regression analyses demonstrated that severity of atherosclerosis and the number of renal cysts were correlated with thoracic aortic circumference, while only the number of renal cysts was correlated with abdominal aortic circumference (p<0.05), which was more predominant in female subjects (p<0.05). Microscopically, significantly more dilated renal tubules (by Students t-test, p<0.05) along with decreased stainability of basement membrane by Periodic acid-Schiff staining and immunostaining of type IV collagen were noted in background renal tissues in cases with numerous renal cysts than in age- and sex-matched controls without renal cysts (n=10 vs. 10). The present study suggests that a syndrome that affects both aorta and renal tubules may exist.

Novel form of miR-29b suppresses bleomycin-induced pulmonary fibrosis.

MicroRNA 29b (miR-29b) replacement therapy is effective for suppressing fibrosis in a mouse model. However, to develop clinical applications for miRNA mimics, the side effects of nucleic acid drugs have to be addressed. In this study, we focused on miRNA mimics in order to develop therapies for idiopathic pulmonary fibrosis. We developed a single-stranded RNA, termed “miR-29b Psh-match,” that has a unique structure to avoid problems associated with the therapeutic uses of miRNAs. A comparison of miR-29b Psh-match and double-stranded one, termed “miR-29b mimic” indicated that the single-stranded form was significantly effective towards fibrosis according to both in vivo and in vitro experiments. This novel form of miR-29b may become the foundation for developing an effective therapeutic drug for pulmonary fibrosis.