Publications by authors named "Toshihiro Takizawa"

75 Publications

Long Noncoding RNA and Transcription Factor HOXB13 Modulate the Expression of Bone Metastasis-Related Genes in Prostate Cancer.

Genes (Basel) 2021 01 27;12(2). Epub 2021 Jan 27.

Department of Molecular Medicine and Anatomy, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan.

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression, which play fundamental roles in cancer development. In this study, we found that , a highly expressed lncRNA in cell lines derived from prostate cancer bone metastases, promoted the cell invasion and proliferation of PC3 prostate cancer cells. Transcription factor homeobox B13 (HOXB13) was identified as an upstream regulator of regulated bone metastasis-associated C-C motif chemokine ligand 2 (CCL2)/C-C chemokine receptor type 2 (CCR2) signaling in both PC3 prostate cancer cells and SaOS2 osteoblastic cells. The HOXB13/ axis also regulated integrin subunits (ITGAV and ITGB1) specific to prostate cancer bone metastasis. HOXB13, in combination with , directly regulated the () promoter. Furthermore, conditioned medium containing secreted from PC3 cells could induce the expression of and in SaOS2 osteoblastic cells. These results suggest that prostate cancer and HOXB13 promote metastasis by regulation of CCL2/CCR2 cytokine and integrin signaling in autocrine and paracrine manners.
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http://dx.doi.org/10.3390/genes12020182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912412PMC
January 2021

LncRNA -Derived Accelerates the Invasion of Extravillous Trophoblast Cells by Inhibiting and Subsequently Activating Matrix Metalloproteinases.

Int J Mol Sci 2021 Jan 27;22(3). Epub 2021 Jan 27.

Department of Molecular Medicine and Anatomy, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan.

The invasion of extravillous trophoblast (EVT) cells into the maternal decidua, which plays a crucial role in the establishment of a successful pregnancy, is highly orchestrated by a complex array of regulatory mechanisms. Non-coding RNAs (ncRNAs) that fine-tune gene expression at epigenetic, transcriptional, and post-transcriptional levels are involved in the regulatory mechanisms of EVT cell invasion. However, little is known about the characteristic features of EVT-associated ncRNAs. To elucidate the gene expression profiles of both coding and non-coding transcripts (i.e., mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs)) expressed in EVT cells, we performed RNA sequencing analysis of EVT cells isolated from first-trimester placentae. RNA sequencing analysis demonstrated that the lncRNA and its derived miRNA were enriched in EVT cells. Although acts as a placental/trophoblast growth suppressor, there is little information on the involvement of in trophoblast cell invasion. Next, we evaluated a possible role of in EVT cell invasion using the EVT cell lines HTR-8/SVneo and HChEpC1b; overexpression of significantly promoted the invasion of both EVT cell lines. The transcription factor gene was shown to be a target of ; moreover, small interfering RNA-mediated knockdown significantly promoted cell invasion. Furthermore, we identified MMP13 and MMP14 as downstream effectors of /-dependent EVT cell invasion. These findings suggest that -mediated inhibition accelerates EVT cell invasion by upregulating matrix metalloproteinases.
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http://dx.doi.org/10.3390/ijms22031237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866107PMC
January 2021

Extravillous trophoblast invasion accelerated by WNT3A, 5A, and 10B via CD44.

J Matern Fetal Neonatal Med 2021 Oct 17;34(20):3377-3385. Epub 2019 Nov 17.

Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo, Japan.

Introduction: Appropriate extravillous trophoblast (EVT) invasion is essential for successful pregnancy. Previously, we showed that EVTs express CD44, which accelerated EVT invasion. However, its regulation mechanism CD44 remains unknown. Our hypothesis was that WNT signaling enhanced EVT invasion CD44. To test this hypothesis, we investigated the effects of WNT ligands on CD44 expression and EVT invasion using EVT cell lines and isolated primary EVTs.

Methods: We used EVT cell lines (HTR8/SVneo and HChEpC1b) and isolated primary EVTs, extracted from first-trimester trophoblasts. The cells were supplemented with WNT3A, 5A, and 10B. We examined cell invasion and the expressions of CD44 and matrix metalloproteinase (MMP) 9. Next, to clarify the pathway of WNT10B in EVTs, we knock-downed WNT10B using siRNA and activated or inhibited the WNT canonical pathway using an activator (lithium chloride) or inhibitor (FH535, XAV939) with WNT10B addition.

Results: WNT3A, 5A, and 10B accelerated the invasion in the EVT lines and isolated primary EVTs. The expressions of CD44 and MMP9 were also upregulated by WNT ligands. WNT10B knockdown significantly inhibited EVT invasion concomitantly with CD44 expression. The WNT canonical pathway activator upregulated CD44 expression and its inhibitor downregulated it with WNT10B addition.

Conclusions: The present study is the first to show the possibility that WNT3A, WNT5A, and WNT10B exist upstream of CD44 in EVTs. Among them, WNT10B may be a novel accelerator of EVT invasion. WNT signaling mediated by multiple WNT ligands may contribute to EVT invasion.
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http://dx.doi.org/10.1080/14767058.2019.1684891DOI Listing
October 2021

1700108J01Rik and 1700101O22Rik are mouse testis-specific long non-coding RNAs.

Histochem Cell Biol 2018 May 6;149(5):517-527. Epub 2018 Feb 6.

Department of Molecular Medicine and Anatomy, Nippon Medical School, 1-1-5 Sendagi, Tokyo, 113-8602, Japan.

Long non-coding RNAs (lncRNAs; > 200 nucleotides in length) have attracted attention as fine-tuners of gene expression. However, little is known about the cell- and stage-specific expression pattern and function of lncRNAs in spermatogenesis. The purpose of this study was to identify mouse testis-associated lncRNAs using a combination of computational and experimental approaches. We first used the FANTOM5 database to survey lncRNA expression in the mouse testis and performed reverse transcription quantitative polymerase chain reaction (real-time PCR) and in situ hybridization (ISH) analyses. In silico analysis showed that most of the highly expressed lncRNAs in the adult mouse testis were testis-specific lncRNAs and were expressed at and following the initiation of spermatogenesis. We selected the antisense lncRNA 1700108J01Rik and long intergenic non-coding RNA 1700101O22Rik from the most highly expressed lncRNAs in the adult testis for further analysis. Real-time PCR analysis confirmed that 1700108J01Rik and 1700101O22Rik were specifically expressed in the testis. ISH analysis revealed that the two mouse-testis-specific lncRNAs were expressed exclusively in testicular germ cells in meiotic prophase and the round spermatid stage, which coincide with the period of transcriptional reactivation during spermatogenesis. The cytoplasmic distribution of these lncRNAs revealed by ISH suggests their involvement in post-transcriptional gene regulation rather than in epigenetic or transcriptional regulation. Our data provide new insight into testis-associated lncRNAs that will be useful in expression and functional studies of spermatogenesis.
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http://dx.doi.org/10.1007/s00418-018-1642-4DOI Listing
May 2018

Expression Profiling of MicroRNAs in the Inner Ear of Elderly People by Real-Time PCR Quantification.

Audiol Neurootol 2017 30;22(3):135-145. Epub 2017 Sep 30.

Department of Otolaryngology, Nippon Medical School, Tokyo, Japan.

The molecular mechanisms underlying age-related hearing loss are unknown, and currently, there is no treatment for this condition. Recent studies have shown that microRNAs (miRNAs) and age-related diseases are intimately linked, suggesting that some miRNAs may present attractive therapeutic targets. In this study, we obtained 8 human temporal bones from 8 elderly subjects at brain autopsy in order to investigate the expression profile of miRNAs in the inner ear with miRNA arrays. A mean of 478 different miRNAs were expressed in the samples, of which 348 were commonly expressed in all 8 samples. Of these, levels of 16 miRNAs significantly differed between young elderly and old elderly subjects. miRNAs, which play important roles in inner ear development, were detected in all samples, i.e., in both young and old elderly subjects, whether with or without hearing loss. Our results suggest that these miRNAs play important roles not only in development, but also in the maintenance of inner ear homeostasis.
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http://dx.doi.org/10.1159/000479724DOI Listing
July 2018

The Complex Structure of the Mouse Placental Labyrinth Revealed by Double Immunofluorescence Labeling of Slc2a1 and Gjb2.

J Nippon Med Sch 2017;84(3):108-109

Department of Molecular Medicine and Anatomy, Graduate School of Medicine, Nippon Medical School.

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http://dx.doi.org/10.1272/jnms.84.108DOI Listing
June 2019

Expression profiles and circulation dynamics of rat mesenteric lymph microRNAs.

Mol Med Rep 2017 Apr 28;15(4):1989-1996. Epub 2017 Feb 28.

Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo 113‑8602, Japan.

Mesenteric lymph is vital for immune cell trafficking and intestinal fluid and chyle transport, which aid homeostatic maintenance. There have been few reports investigating the profiles and circulatory dynamics of mesenteric lymph microRNAs (miRNAs). The present study aimed to provide a comprehensive analysis of miRNAs in normal rodent mesenteric lymph. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR)‑based array analysis was performed to examine the expression levels of 375 miRNAs in normal rat mesenteric lymph. Using differential centrifugation, the presence of miR‑150, a representative lymph miRNA, in exosomes was assessed. Rat small intestine epithelial cell line IEC‑6‑derived exosomes were prepared from culture supernatants of cells transfected with cel‑miR‑238‑3p, and were used to trace the administered exosomes in vivo and to investigate the in vivo delivery of lymph miRNAs via mesenteric lymphatics into the systemic circulation following injection of cel‑miR‑238‑3p‑exosomes. RT‑qPCR‑based array analysis detected 287 miRNAs in lymph, and 21 miRNAs that were significantly differentially expressed between lymph and plasma. Lymph fractionation analysis demonstrated that some cell‑free lymph miR‑150 was distributed in the exosome‑containing microsomal fraction. Furthermore, in vivo analysis of lymph miRNA delivery revealed that exosomal cel‑miR‑238‑3p was markedly distributed in the lung compared with in the liver, kidney and spleen, thus indicating that the lung is the major organ responsible for clearance of exosomal lymph miRNAs. These findings provide novel insights into the modulation of gene expression by mesenteric lymph miRNAs in the lung.
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http://dx.doi.org/10.3892/mmr.2017.6259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365009PMC
April 2017

Endogenous and exogenous miR-520c-3p modulates CD44-mediated extravillous trophoblast invasion.

Placenta 2017 02 14;50:25-31. Epub 2016 Dec 14.

Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo 113-8602, Japan. Electronic address:

Introduction: Adequate extravillous trophoblast (EVT) invasion is essential for successful placentation. Although miR-520c-3p plays an important role in CD44-mediated invasion in cancer cells, there is little information on whether miR-520c-3p is involved in the regulatory mechanisms of CD44-mediated EVT invasion.

Methods: We screened first trimester trophoblast cells and trophoblast cell lines for expression of miR-520c-3p using real-time polymerase chain reaction. The cell invasion assay was performed using EVT cell lines, HTR8/SVneo and HChEpC1b, to investigate the capability of suppressing EVT invasion by miR-520c-3p. Laser microdissection analysis was then used to determine whether miR-520c-3p was present in the first trimester decidua. Finally, the possibility of chorionic villous trophoblast (CVT)-EVT communication via exosomal miR-520c-3p was determined using an in vitro model based on BeWo exosomes and the EVT cell lines as recipient cells.

Results: The miR-520c-3p level was significantly downregulated in EVT cell lines and EVTs. Cell invasion was significantly inhibited in miR-520c-3p-overexpressing cell lines, involving a significant reduction of CD44. Laser microdissection analysis showed that miR-520c-3p in the periarterial area of the decidua was significantly higher than that in the non-periarterial area. Using an in vitro model system, BeWo exosomal miR-520c-3p was internalized into the EVT cells with subsequently reduced cell invasion via CD44 repression.

Conclusions: EVT invasion is synergistically enhanced by the reciprocal expression of endogenous miR-520c-3p and CD44. The present study supports a novel model involving a placenta-associated miRNA function in cell-cell communication in which CVT exosomal miR-520c-3p regulates cell invasion by targeting CD44 in EVTs.
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http://dx.doi.org/10.1016/j.placenta.2016.12.016DOI Listing
February 2017

Wnt5a and preeclampsia: is Wnt5a really a causative factor of preeclampsia?

J Matern Fetal Neonatal Med 2017 May 4;30(9):1087-1088. Epub 2016 Jul 4.

a Department of Obstetrics and Gynecology , Jichi Medical University , Shimotsuke , Japan and.

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http://dx.doi.org/10.1080/14767058.2016.1203412DOI Listing
May 2017

FluoroNanogold: an important probe for correlative microscopy.

J Chem Biol 2015 Oct 25;8(4):129-42. Epub 2015 Aug 25.

Department of Physiology and Cell Biology, Ohio State University, Columbus, OH 43210 USA.

Correlative microscopy is a powerful imaging approach that refers to observing the same exact structures within a specimen by two or more imaging modalities. In biological samples, this typically means examining the same sub-cellular feature with different imaging methods. Correlative microscopy is not restricted to the domains of fluorescence microscopy and electron microscopy; however, currently, most correlative microscopy studies combine these two methods, and in this review, we will focus on the use of fluorescence and electron microscopy. Successful correlative fluorescence and electron microscopy requires probes, or reporter systems, from which useful information can be obtained with each of the imaging modalities employed. The bi-functional immunolabeling reagent, FluoroNanogold, is one such probe that provides robust signals in both fluorescence and electron microscopy. It consists of a gold cluster compound that is visualized by electron microscopy and a covalently attached fluorophore that is visualized by fluorescence microscopy. FluoroNanogold has been an extremely useful labeling reagent in correlative microscopy studies. In this report, we present an overview of research using this unique probe.
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http://dx.doi.org/10.1007/s12154-015-0145-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744603PMC
October 2015

Dysregulated miRNA in progression of hepatocellular carcinoma: A systematic review.

Hepatol Res 2016 Mar 12;46(5):391-406. Epub 2015 Nov 12.

Department of Surgery, Nippon Medical School Hospital, Tokyo, Japan.

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer and the third cause of cancer-related mortality worldwide. The primary risk factor for HCC is liver cirrhosis secondary to persistent infection with hepatitis B virus or hepatitis C virus. Although a number of cellular phenomena and molecular events have been reported to facilitate tumor initiation, progression and metastasis, the exact etiology of HCC has not yet been fully uncovered. miRNA, a class of non-coding RNA, negatively regulate post-transcriptional processes that participate in crucial biological processes, including development, differentiation, apoptosis and proliferation. In the liver, specific miRNA can be negative regulators of gene expression. Recent studies have uncovered the contribution of miRNA to cancer pathogenesis as they can function as oncogenes or tumor suppressor genes. In addition, other studies have demonstrated their potential value in the clinical management of patients with HCC as some miRNA may be used as prognostic or diagnostic markers. In this review, we summarize the current knowledge about the roles of miRNA in the carcinogenesis and progression of HCC.
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http://dx.doi.org/10.1111/hepr.12606DOI Listing
March 2016

Host cellular microRNA involvement in the control of hepatitis B virus gene expression and replication.

World J Hepatol 2015 Apr;7(4):696-702

Yoshiaki Mizuguchi, Eiji Uchida, Department of Surgery, Nippon Medical School Hospital, Bunkyo-Ku, Tokyo 113-8603, Japan.

A large number of studies have demonstrated that the synergistic collaboration of a number of microRNAs (miRNAs), their growth factors and their downstream agents is required for the initiation and completion of pathogenesis in the liver. miRNAs are thought to exert a profound effect on almost every aspect of liver biology and pathology. Accumulating evidence indicates that several miRNAs are involved in the hepatitis B virus (HBV) life cycle and infectivity, in addition to HBV-associated liver diseases including fibrosis, cirrhosis and hepatocellular carcinoma (HCC). In turn, HBV can modulate the expression of several cellular miRNAs, thus promoting a favorable environment for its replication and survival. In this review, we focused on the involvement of host cellular miRNAs that are directly and indirectly associated with HBV RNA or HBV associated transcription factors. Exploring different facets of the interactions among miRNA, HBV and HCV infections, and the carcinogenesis and progress of HCC, could facilitate the development of novel and effective treatment approaches for liver disease.
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http://dx.doi.org/10.4254/wjh.v7.i4.696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4388997PMC
April 2015

Maternal peripheral blood natural killer cells incorporate placenta-associated microRNAs during pregnancy.

Int J Mol Med 2015 Jun 27;35(6):1511-24. Epub 2015 Mar 27.

Department of Molecular Medicine and Anatomy, Graduate School of Medicine, Nippon Medical School, Tokyo 113‑8602, Japan.

Although recent studies have demonstrated that microRNAs (miRNAs or miRs) regulate fundamental natural killer (NK) cellular processes, including cytotoxicity and cytokine production, little is known about the miRNA-gene regulatory relationships in maternal peripheral blood NK (pNK) cells during pregnancy. In the present study, to determine the roles of miRNAs within gene regulatory networks of maternal pNK cells, we performed comprehensive miRNA and gene expression profiling of maternal pNK cells using a combination of reverse transcription quantitative PCR (RT-qPCR)-based miRNA array and DNA microarray analyses and analyzed the differential expression levels between first- and third-trimester pNK cells. Furthermore, we constructed regulatory networks for miRNA-mediated gene expression in pNK cells during pregnancy by Ingenuity Pathway Analysis (IPA). PCR-based array analysis revealed that the placenta-derived miRNAs [chromosome 19 miRNA cluster (C19MC) miRNAs] were detected in pNK cells during pregnancy. Twenty-five miRNAs, including six C19MC miRNAs, were significantly upregulated in the third- compared to first-trimester pNK cells. The rapid clearance of C19MC miRNAs also occurred in the pNK cells following delivery. Nine miRNAs, including eight C19MC miRNAs, were significantly downregulated in the post-delivery pNK cells compared to those of the third-trimester. DNA microarray analysis identified 69 NK cell function-related genes that were differentially expressed between the first- and third-trimester pNK cells. On pathway and network analysis, the observed gene expression changes of pNK cells likely contribute to the increase in the cytotoxicity, as well as the cell cycle progression of third- compared to first-trimester pNK cells. Thirteen of the 69 NK cell function-related genes were significantly downregulated between the first- and third-trimester pNK cells. Nine of the 13 downregulated NK-function-associated genes were in silico target candidates of 12 upregulated miRNAs, including C19MC miRNA miR-512-3p. The results of this study suggest that the transfer of placental C19MC miRNAs into maternal pNK cells occurs during pregnancy. The present study provides new insight into maternal NK cell functions.
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http://dx.doi.org/10.3892/ijmm.2015.2157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432927PMC
June 2015

Sex differences in shotgun proteome analyses for chronic oral intake of cadmium in mice.

PLoS One 2015 20;10(3):e0121819. Epub 2015 Mar 20.

Isotope Research Center, Nippon Medical School, Bunkyo-ku, Tokyo, Japan.

Environmental diseases related to cadmium exposure primarily develop owing to industrial wastewater pollution and/or contaminated food. In regions with high cadmium exposure in Japan, cadmium accumulation occurs primarily in the kidneys of individuals who are exposed to the metal. In contrast, in the itai-itai disease outbreak that occurred in the Jinzu River basin in Toyama Prefecture in Japan, cadmium primarily accumulated in the liver. On the other hand, high concentration of cadmium caused renal tubular disorder and osteomalacia (multiple bone fracture), probably resulting from the renal tubular dysfunction and additional pathology. In this study, we aimed to establish a mouse model of chronic cadmium intake. We administered cadmium-containing drinking water (32 mg/l) to female and male mice ad libitum for 11 weeks. Metal analysis using inductively coupled plasma mass spectrometry revealed that cadmium accumulated in the kidneys (927 x 10 + 185 ng/g in females and 661 x 10 + 101 ng/g in males), liver (397 x 10 + 199 ng/g in females and 238 x 10 + 652 ng/g in males), and thyroid gland (293 + 93.7 ng/g in females and 129 + 72.7 ng/g in males) of mice. Female mice showed higher cadmium accumulation in the kidney, liver, and thyroid gland than males did (p = 0.00345, p = 0.00213, and p = 0.0331, respectively). Shotgun proteome analyses after chronic oral administration of cadmium revealed that protein levels of glutathione S-transferase Mu2, Mu4, and Mu7 decreased in the liver, and those of A1 and A2 decreased in the kidneys in both female and male mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121819PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368563PMC
February 2016

Fc gamma receptor IIb participates in maternal IgG trafficking of human placental endothelial cells.

Int J Mol Med 2015 May 17;35(5):1273-89. Epub 2015 Mar 17.

Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo 113-8602, Japan.

The human placental transfer of maternal IgG is crucial for fetal and newborn immunity. Low-affinity immunoglobulin gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (the FCGR2B2 compartment) in human placental endothelial cells; thus, we hypothesized that the FCGR2B2 compartment functions as an IgG transporter. In this study, to examine this hypothesis, we performed in vitro bio-imaging analysis of IgG trafficking by FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced GFP-tagged FCGR2B2 (pFCGR2B2-EGFP). FCGR2B2-EGFP signals were detected as intracellular vesicular structures similar to FCGR2B2 compartments in vivo. The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments. Among the RABs, RAB3D was expressed predominantly in placental endothelial cells. The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery. Taken together, these findings suggest that FCGR2B2 compartments participate in the transcytosis of maternal IgG across the human placental endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments.
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http://dx.doi.org/10.3892/ijmm.2015.2141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380207PMC
May 2015

Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia.

J Hepatol 2015 Aug 12;63(2):399-407. Epub 2015 Mar 12.

Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, PA 15260, USA; The Department of Pathology, Division of Liver and Transplantation Pathology, University of Pittsburgh Medical Center, PA 15260, USA. Electronic address:

Background & Aims: Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers.

Methods: Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a.

Results: BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers.

Conclusion: BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment.
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http://dx.doi.org/10.1016/j.jhep.2015.02.047DOI Listing
August 2015

BRAF, KIT and NRAS mutations and expression of c-KIT, phosphorylated extracellular signal-regulated kinase and phosphorylated AKT in Japanese melanoma patients.

J Dermatol 2015 May 13;42(5):477-84. Epub 2015 Mar 13.

Department of Dermatology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.

To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c-KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma.
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http://dx.doi.org/10.1111/1346-8138.12822DOI Listing
May 2015

Human exosomal placenta-associated miR-517a-3p modulates the expression of PRKG1 mRNA in Jurkat cells.

Biol Reprod 2014 Nov 1;91(5):129. Epub 2014 Oct 1.

Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo, Japan

During pregnancy, human placenta-associated microRNAs (miRNAs) derived from the miRNA cluster in human chromosome 19 are expressed in villous trophoblasts and secreted into maternal circulation via exosomes; however, little is known about whether circulating placenta-associated miRNAs are transferred into maternal immune cells via exosomes, and modulate expression of target genes in the recipient cells. We employed an in vitro model of trophoblast-immune cell communication using BeWo cells (a human trophoblast cell line) and Jurkat cells (a human leukemic T-cell line) and investigated whether BeWo exosomal placenta-associated miRNAs can suppress expression of target genes in the recipient Jurkat cells. Using this system, we identified PRKG1 as a target gene of placenta-associated miRNA miR-517a-3p. Moreover, we demonstrated that BeWo exosomal miR-517a-3p was internalized into Jurkat cells and subsequently suppressed the expression of PRKG1 in recipient Jurkat cells. Furthermore, using peripheral blood natural killer (NK) cells in vivo, we confirmed that circulating miR-517a-3p was delivered into maternal NK cells as it was into Jurkat cells in vitro. Placenta-associated miR-517a-3p was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. Expression levels of miR-517a-3p and its target mRNA PRKG1 were inversely correlated in NK cells before and after delivery. These in vitro and in vivo results suggest that exosome-mediated transfer of placenta-associated miRNAs and subsequent modulation of their target genes occur in maternal NK cells. The present study provides novel insight into our understanding of placenta-maternal communication.
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http://dx.doi.org/10.1095/biolreprod.114.121616DOI Listing
November 2014

Differential expression of ADAM (a disintegrin and metalloproteinase) genes between human first trimester villous and extravillous trophoblast cells.

J Nippon Med Sch 2014 ;81(3):122-9

Department of Obstetrics and Gynecology, Jichi Medical University.

A disintegrin and metalloproteinases (ADAMs) are members of the metzincin family of zinc-dependent metalloproteinases that play pivotal roles in the proteolytic degradation of the extracellular matrix for cell invasion. Few studies have investigated the ADAM subtypes that are expressed in first trimester trophoblast cells. The purpose of this study was to elucidate the differential expression profiles of ADAMs between first trimester villous trophoblast cells (VTs) and extravillous trophoblast cells (EVTs). We isolated EVTs from explanted human first trimester chorionic villi and investigated the mRNA expression levels of five members of the ADAM family (ADAMTS1, ADAMTS2, ADAM10, ADAM12, and ADAM17) using real-time PCR. Chorionic villous tips were defined as first trimester VTs. Of the differentially expressed ADAM genes between first trimester VTs and EVTs, ADAMTS1 was expressed at a significantly higher level in EVTs than in VTs. In contrast, both ADAM10 and ADAM12 were expressed at significantly higher levels in VTs than in EVTs. No differences were found in the mRNA levels of ADAMTS2 and ADAM17 between the two cell types. Moreover, we demonstrated that in VTs, the expression level of ADAM12 was significantly downregulated in the late first trimester (10-13 gestational weeks) compared to the middle first trimester (7-8 weeks). These results suggest that first trimester trophoblast cells express ADAM genes in cell type- and gestational age-dependent manners. Our data provide additional insight into the functions of ADAMs in the human placenta.
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http://dx.doi.org/10.1272/jnms.81.122DOI Listing
April 2015

Sevoflurane anesthesia persistently downregulates muscle-specific microRNAs in rat plasma.

Int J Mol Med 2014 Jul 9;34(1):291-8. Epub 2014 Apr 9.

Department of Molecular Medicine and Anatomy, Graduate School of Medicine, Nippon Medical School, Tokyo 113-8602, Japan.

The volatile anesthetic, sevoflurane, is widely used in surgery. Over the years, there has been a growing interest in the biological effects of sevoflurane on tissue and organ systems and the molecular mechanisms involved. MicroRNAs (miRNAs or miRs) acting as pivotal post‑transcriptional regulators for fine-tuning gene networks are not only expressed intracellularly, but are also secreted into the plasma. However, the sevoflurane‑associated dynamics of circulating miRNAs and the effects of sevoflurane on tissues remain unknown. Thus, the aim of this study was to perform a comprehensive analysis of circulating miRNA levels and compositions in sevoflurane‑anesthetized rats. The rats were allowed to breathe spontaneously under 2% sevoflurane anesthesia for 6 h, and we performed a quantitative polymerase chain reaction (PCR)‑based array analysis of the time-dependent changes in plasma miRNA levels and compositions. Subsequently, we validated the levels of muscle‑specific miRNAs (also known as myomiRNAs; miR-1, miR‑133a, miR-133b and miR-206) of the plasma, heart and skeletal muscle by quantitative PCR following 3 and 6 h of anesthesia, as well as at 1, 3, 7 and 14 days post-anesthesia. Of the 210 miRNAs detected in the rat plasma from the control group (no anesthesia), 161 plasma miRNAs (77%) were transiently downregulated as a result of sevoflurane anesthesia. Although the downregulation of the plasma miRNAs (148 out of the 161 plasma mRNAs; 92%) recovered immediately after anesthesia, the plasma levels of 4 muscle-specific miRNAs were persistently downregulated until 14 days post-anesthesia. In the cardiac and skeletal muscles, the expression levels of the muscle-specific miRNAs were upregulated within 2 weeks post-anesthesia, indicating that the expression levels of the muscle-specific miRNAs in the cardiac and skeletal muscles and their plasma levels are substantially inversely correlated following anesthesia. Our data suggest that sevoflurane predominantly affects cardiac and skeletal muscles and suppresses the release of miRNA from these tissues into the circulation. This new information provides novel insight into the molecular mechanisms of action of the anesthetic, sevoflurane.
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http://dx.doi.org/10.3892/ijmm.2014.1739DOI Listing
July 2014

Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells.

Nucleic Acids Res 2014 Apr 20;42(8):5256-69. Epub 2014 Feb 20.

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, Department of Biotechnology, Faculty of Engineering, Maebashi Institute of Technology, 460-1 Kamisadori-cho, Maebashi-shi, Gunma 371-0816, Japan and Department of Molecular Medicine and Anatomy, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.

Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5' proximal nucleotides, which include seed sequence (positions 2-8 from the 5' end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the trans-activation response RNA-binding protein (TRBP). The terminal RNA (positions 19-21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15-18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.
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http://dx.doi.org/10.1093/nar/gku153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005638PMC
April 2014

Changes in expression of vascular endothelial growth factor D-related genes in placental mesenchymal dysplasia.

J Obstet Gynaecol Res 2014 Apr 15;40(4):1145-9. Epub 2014 Jan 15.

Department of Obstetrics and Gynecology, Jichi Medical University, Tochigi, Japan.

A recent report indicated that vascular endothelial growth factor (VEGF)-D, regulating cell proliferation and/or differentiation, may be associated with the development of placental mesenchymal dysplasia (PMD), a disorder characterized by cell proliferation/differentiation. In PMD placenta, we examined the expression of five cell-proliferation/differentiation-associated genes, namely, Wnt3a, Wnt5a, β-catenin, VEGF-D and Dickkopf-1 (DKK-1). In PMD, expressions of Wnt3a, Wnt5a and β-catenin were decreased, whereas those of VEGF-D and DKK-1 were increased. These abnormal expressions suggest a relationship between these genes and PMD pathogenesis/pathophysiology.
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http://dx.doi.org/10.1111/jog.12291DOI Listing
April 2014

Placenta-specific miRNA (miR-512-3p) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells.

J Obstet Gynaecol Res 2014 Mar 18;40(3):650-60. Epub 2013 Nov 18.

Department of Molecular Medicine and Anatomy, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan; Department of Reproductive Medicine, Perinatology and Gynecologic Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.

Aim: The microRNAs (miRNAs) derived from the chromosome 19 miRNA cluster (C19MC) are exclusively expressed in the human placenta, but the origin and functions of C19MC miRNAs are not fully understood. The purpose of this study was to elucidate which cells express C19MC miRNAs in chorionic villi and identify their miRNA targets.

Methods: A combination of laser microdissection (LMD) and real-time polymerase chain reaction (PCR) to examine the localization of five C19MC miRNAs (i.e. miR-512-3p, miR-518b, miR-520a, miR-524 and miR-1323) in the human placenta was performed. Furthermore, to identify miR-512-3p-target genes, we analyzed gene expression profiles of the trophoblast cell line BeWo using a DNA microarray. Predicted target genes were validated by real-time PCR, western blotting, and 3'-untranslated region reporter assay.

Results: By LMD and subsequent PCR analysis, five C19MC miRNAs examined in this study were predominantly expressed in villous trophoblast cells; little expression, if any, was observed in villous stroma cells or fetal endothelial cells. Microarray data showed that 334 genes were downregulated in BeWo cells treated with Pre-miR-512-3p (mature miR-512-3p mimic). We found six candidate target genes of miR-512-3p using DNA microarray data and target prediction software. Furthermore, we revealed that protein phosphatase 3, regulatory subunit B, alpha (PPP3R1), one of the six genes, was a miR-512-3p target using an in vitro experimental validation system.

Conclusion: These data suggest that miR-512-3p participates in human trophoblast function[s] by targeting PPP3R1, encoding a regulatory subunit of calcineurin.
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http://dx.doi.org/10.1111/jog.12217DOI Listing
March 2014

Small proline rich protein 2a in benign and malignant liver disease.

Hepatology 2014 Mar 27;59(3):1130-43. Epub 2014 Jan 27.

Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA; Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA.

Unlabelled: STAT3-driven expression of small proline rich protein 2a (SPRR2a), which acts as an src homology 3 (SH3) domain ligand, induces biliary epithelial cell (BEC) epithelial-mesenchymal transition (EMT), which, in turn, promotes wound healing. SPRR2a also quenches free radicals and protects against oxidative stress and DNA damage in nonneoplastic BEC. Sprr2a-induced EMT also increases local invasiveness of cholangiocarcinomas (CC), but prevents metastases. Understanding SPRR2a regulation of EMT has potential for therapeutic targeting in both benign and malignant liver disease. Molecular mechanisms responsible for SPRR2a-induced EMT were characterized, in vitro, and then evidence for utilization of these pathways was sought in human intrahepatic CC, in vivo, using multiplex labeling and software-assisted morphometric analysis. SPRR2a complexes with ZEB1 and CtBP on the microRNA (miR)-200c/141 promoter resulting in synergic suppression of miR-200c/141 transcription, which is required for maintenance of the BEC epithelial phenotype. SPRR2a induction promotes dephosphorylation and nuclear translocation of the SH3-domain containing protein GRB2 and an SH3-domain ligand in ZEB1 is required for SPRR2a-induced synergic suppression of miR-200c/141. Multiplex protein labeling of CC and morphometric analyses showed: 1) up-regulation of ZEB-1, and 2) down-regulation of CK19 in intrahepatic CC compared to nonneoplastic BEC, consistent with previous CC proteomic studies showing EMT during cholangiocarcinogenesis.

Conclusion: SPRR2a modulates ZEB-1 signaling by way of miR-200c/141-associated EMT through SH3-domain networks and contributes to benign and malignant BEC wound-healing responses.
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http://dx.doi.org/10.1002/hep.26889DOI Listing
March 2014

MiR-376c down-regulation accelerates EGF-dependent migration by targeting GRB2 in the HuCCT1 human intrahepatic cholangiocarcinoma cell line.

PLoS One 2013 26;8(7):e69496. Epub 2013 Jul 26.

Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo, Japan.

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3'-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069496PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724868PMC
April 2014

Combination of protein coding and noncoding gene expression as a robust prognostic classifier in stage I lung adenocarcinoma.

Cancer Res 2013 Jul 2;73(13):3821-32. Epub 2013 May 2.

Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.

Prognostic tests for patients with early-stage lung cancer may provide needed guidance on postoperative surveillance and therapeutic decisions. We used a novel strategy to develop and validate a prognostic classifier for early-stage lung cancer. Specifically, we focused on 42 genes with roles in lung cancer or cancer prognosis. Expression of these biologically relevant genes and their association with relapse-free survival (RFS) were evaluated using microarray data from 148 patients with stage I lung adenocarcinoma. Seven genes associated with RFS were further examined by quantitative reverse transcription PCR in 291 lung adenocarcinoma tissues from Japan, the United States, and Norway. Only BRCA1, HIF1A, DLC1, and XPO1 were each significantly associated with prognosis in the Japan and US/Norway cohorts. A Cox regression-based classifier was developed using these four genes on the Japan cohort and validated in stage I lung adenocarcinoma from the US/Norway cohort and three publicly available lung adenocarcinoma expression profiling datasets. The results suggest that the classifier is robust across ethnically and geographically diverse populations regardless of the technology used to measure gene expression. We evaluated the combination of the four-gene classifier with miRNA miR-21 (MIR21) expression and found that the combination improved associations with prognosis, which were significant in stratified analyses on stage IA and stage IB patients. Thus, the four coding gene classifier, alone or with miR-21 expression, may provide a clinically useful tool to identify high-risk patients and guide recommendations regarding adjuvant therapy and postoperative surveillance of patients with stage I lung adenocarcinoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-0031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503978PMC
July 2013

The miR-221/222 cluster, miR-10b and miR-92a are highly upregulated in metastatic minimally invasive follicular thyroid carcinoma.

Int J Oncol 2013 Jun 2;42(6):1858-68. Epub 2013 Apr 2.

Department of Molecular Medicine and Anatomy, Nippon Medical School, Tokyo, Japan.

Minimally invasive follicular thyroid carcinoma (MI-FTC) is characterized by limited capsular and/or vascular invasion with good long-term outcomes. However, some cases of MI-FTC show a poor prognosis because of severe distant metastasis (i.e., metastatic MI-FTC). Nonetheless, no method has been established for predicting the prognosis of MI-FTC. This study was conducted to identify novel prognostic factors for metastatic MI-FTC by the use of microRNA (miRNA). Thirty-four patients with MI-FTC were categorized into two groups: the metastatic group, M(+) (n=12) and the non-metastatic group, M(-) (n=22). In the M(+) group, distant metastasis was recognized after the initial operation established the diagnosis of MI-FTC. In the M(-) group, no distant metastasis was recognized postoperatively for ≥ 10 years. Using laser microdissection followed by quantitative real-time PCR and PCR arrays, we performed a comprehensive expression profiling of 667 miRNAs in formalin-fixed, paraffin-embedded samples from the initial MI-FTC operation. Furthermore, we assessed the potential use of miRNAs as novel biomarkers for the metastatic potential of MI-FTC by logistic regression analysis. Comprehensive quantitative analysis of miRNA expression in MI-FTC samples revealed that the miR-221/222 cluster (i.e., miR-221, miR-222 and miR-222*), miR-10b and miR-92a were significantly upregulated in the M(+) group compared with the M(-) group. Interestingly, the expression levels of these miRNAs were also shown to be upregulated in widely invasive FTC (WI-FTC; n=13) that has distant metastasis and worse prognosis, indicating a close similarity in the miRNA expression between metastatic MI-FTC and WI-FTC. Logistic regression analysis revealed that miR-10b made a significant contribution to prognosis (OR 19.759, 95% CI 1.433-272.355, p=0.026). Our findings suggest that miR-10b is a potential prognostic factor for evaluating the metastatic potential of MI-FTC at an initial operation stage.
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http://dx.doi.org/10.3892/ijo.2013.1879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699596PMC
June 2013

SnoN/SKIL modulates proliferation through control of hsa-miR-720 transcription in esophageal cancer cells.

Biochem Biophys Res Commun 2013 Jan 12;430(1):101-6. Epub 2012 Nov 12.

Department of Surgery for Organ Function and Biological Regulation, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-Ku, Tokyo 113-8602, Japan.

It is now evident that changes in microRNA are involved in cancer progression, but the mechanisms of transcriptional regulation of miRNAs remain unknown. Ski-related novel gene (SnoN/SKIL), a transcription co-factor, acts as a potential key regulator within a complex network of p53 transcriptional repressors. SnoN has pro- and anti-oncogenic functions in the regulation of cell proliferation, senescence, apoptosis, and differentiation. We characterized the roles of SnoN in miRNA transcriptional regulation and its effects on cell proliferation using esophageal squamous cell carcinoma (ESCC) cells. Silencing of SnoN altered a set of miRNA expression profiles in TE-1cells, and the expression levels of miR-720, miR-1274A, and miR-1274B were modulated by SnoN. The expression of these miRNAs resulted in changes to the target protein p63 and a disintegrin and metalloproteinase domain 9 (ADAM9). Furthermore, silencing of SnoN significantly upregulated cell proliferation in TE-1 cells, indicating a potential anti-oncogenic function. These results support our observation that cancer tissues have lower expression levels of SnoN, miR-720, and miR-1274A compared to adjacent normal tissues from ESCC patients. These data demonstrate a novel mechanism of miRNA regulation, leading to changes in cell proliferation.
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http://dx.doi.org/10.1016/j.bbrc.2012.11.005DOI Listing
January 2013

Expression of flotillins in the human placenta: potential implications for placental transcytosis.

Histochem Cell Biol 2013 Mar 14;139(3):487-500. Epub 2012 Oct 14.

Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

A proteomics survey of human placental syncytiotrophoblast (ST) apical plasma membranes revealed peptides corresponding to flotillin-1 (FLOT1) and flotillin-2 (FLOT2). The flotillins belong to a class of lipid microdomain-associated integral membrane proteins that have been implicated in clathrin- and caveolar-independent endocytosis. In the present study, we characterized the expression of the flotillin proteins within the human placenta. FLOT1 and FLOT2 were coexpressed in placental lysates and BeWo human trophoblast cells. Immunofluorescence microscopy of first-trimester and term placentas revealed that both proteins were more prominent in villous endothelial cells and cytotrophoblasts (CTs) than the ST. Correspondingly, forskolin-induced fusion in BeWo cells resulted in a decrease in FLOT1 and FLOT2, suggesting that flotillin protein expression is reduced following trophoblast syncytialization. The flotillin proteins co-localized with a marker of fluid-phase pinocytosis, and knockdown of FLOT1 and/or FLOT2 expression resulted in decreased endocytosis of cholera toxin B subunit. We conclude that FLOT1 and FLOT2 are abundantly coexpressed in term villous placental CTs and endothelial cells, and in comparison, expression of these proteins in the ST is reduced. These findings suggest that flotillin-dependent endocytosis is unlikely to be a major pathway in the ST, but may be important in the CT and endothelium.
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http://dx.doi.org/10.1007/s00418-012-1040-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574205PMC
March 2013

Correlative fluorescence and transmission electron microscopy in tissues.

Methods Cell Biol 2012 ;111:37-57

Department of Molecular Anatomy, Nippon Medical School, Tokyo 113-8602, Japan.

Correlative microscopy has meant different things over the years; currently, this term refers to imaging the same exact structures with two or more imaging modalities. This commonly involves combining fluorescence and electron microscopy. Much of the recent work related to correlative microscopy has been done using cell culture models. However, many biological questions cannot be addressed in these models, but require instead the 3-dimensional organization of cells found in tissues. Herein, we discuss some of the issues related to correlative microscopy of tissues including the major reporter systems presently available for correlative microscopy. We present data from our own work in which we have focused on the use of ultrathin cryosections of tissues as the substrate for immunolabeling to combine immunofluorescence and electron microscopy of the same sub-cellular structures.
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http://dx.doi.org/10.1016/B978-0-12-416026-2.00003-0DOI Listing
December 2012
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