Publications by authors named "Takahiro Kishikawa"

41 Publications

Mutant KRAS drives metabolic reprogramming and autophagic flux in premalignant pancreatic cells.

Cancer Gene Ther 2021 Apr 8. Epub 2021 Apr 8.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan.

Mutational activation of the KRAS gene occurs in almost all pancreatic ductal adenocarcinoma (PDAC) and is the earliest molecular event in their carcinogenesis. Evidence has accumulated of the metabolic reprogramming in PDAC, such as amino acid homeostasis and autophagic flux. However, the biological effects of KRAS mutation on metabolic reprogramming at the earlier stages of PDAC carcinogenesis are unclear. Here we report dynamic metabolic reprogramming in immortalized human non-cancerous pancreatic ductal epithelial cells, in which a KRAS mutation was induced by gene-editing, which may mimic early pancreatic carcinogenesis. Similar to the cases of PDAC, KRAS gene mutation increased the dependency on glucose and glutamine for maintaining the intracellular redox balance. In addition, the intracellular levels of amino acids were significantly decreased because of active protein synthesis, and the cells required greater autophagic flux to maintain their viability. The lysosomal inhibitor chloroquine significantly inhibited cell proliferation. Therefore, metabolic reprogramming is an early event in carcinogenesis initiated by KRAS gene mutation, suggesting a rationale for the development of nutritional interventions that suppress or delay the development of PDAC.
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http://dx.doi.org/10.1038/s41417-021-00326-4DOI Listing
April 2021

Post-treatment cell-free DNA as a predictive biomarker in molecular-targeted therapy of hepatocellular carcinoma.

J Gastroenterol 2021 May 12;56(5):456-469. Epub 2021 Mar 12.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Background: Liquid biopsies, particularly those involving circulating tumor DNA (ctDNA), are rapidly emerging as a non-invasive alternative to tumor biopsies. However, clinical applications of ctDNA analysis in hepatocellular carcinoma (HCC) have not been fully elucidated.

Methods: We measured the amount of plasma-derived cell-free DNA (cfDNA) in HCC patients before (n = 100) and a few days after treatment (n = 87), including radiofrequency ablation, transarterial chemoembolization, and molecular-targeted agents (MTAs), and prospectively analyzed their associations with clinical parameters and prognosis. TERT promoter mutations in cfDNA were analyzed using droplet digital PCR. Furthermore, we performed a comprehensive mutational analysis of post-treatment cfDNA via targeted ultra-deep sequencing (22,000× coverage) in a panel of 275 cancer-related genes in selected patients.

Results: Plasma cfDNA levels increased significantly according to HCC clinical stage, and a high cfDNA level was independently associated with a poor prognosis. TERT promoter mutations were detected in 45% of all cases but were not associated with any clinical characteristics. cfDNA levels increased significantly a few days after treatment, and a greater increase in post-treatment cfDNA levels was associated with a greater therapeutic response to MTAs. The detection rate of TERT mutations increased to 57% using post-treatment cfDNA, suggesting that the ctDNA was enriched. Targeted ultra-deep sequencing using post-treatment cfDNA after administering lenvatinib successfully detected various gene mutations and obtained promising results in lenvatinib-responsive cases.

Conclusions: Post-treatment cfDNA analysis may facilitate the construction of biomarkers for predicting MTA treatment effects.
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http://dx.doi.org/10.1007/s00535-021-01773-4DOI Listing
May 2021

WWP1 Gain-of-Function Inactivation of PTEN in Cancer Predisposition.

N Engl J Med 2020 05;382(22):2103-2116

From the Cancer Research Institute, Beth Israel Deaconess Cancer Center (Y.-R.L., T.K., J.Z., N.P., J.L., W.W., P.P.P.), and the Departments of Medicine (Y.-R.L., T.K., N.P., P.P.P.) and Pathology (J.Z., J.L., W.W., P.P.P.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston; the Genomic Medicine Institute (L.Y., Y.N., B.L., C.E.) and the Department of Quantitative Health Sciences (Y.N.), Lerner Research Institute, Cleveland Clinic, the Taussig Cancer Institute (C.E.), the Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine (C.E.), and the Germline High Risk Cancer Focus Group, Case Comprehensive Cancer Center, Case Western Reserve University (C.E.) - all in Cleveland; the Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University (J.Z.), and the Medical Research Institute, Wuhan University (J.Z.) - both in Wuhan, China; and the Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy (P.P.P.).

Background: Patients with hamartoma tumor syndrome (PHTS) have germline mutations in the tumor-suppressor gene encoding phosphatase and tensin homologue (). Such mutations have been associated with a hereditary predisposition to multiple types of cancer, including the Cowden syndrome. However, a majority of patients who have PHTS-related phenotypes have tested negative for mutations. In a previous study, we found that the E3 ubiquitin ligase WWP1 negatively regulates the function of PTEN.

Methods: In a prospective cohort study conducted from 2005 through 2015, we enrolled 431 patients with wild-type who met at least the relaxed diagnostic criteria of the International Cowden Consortium. Patients were scanned for germline variants. We used the Cancer Genome Atlas (TCGA) data set as representative of apparently sporadic cancers and the Exome Aggregation Consortium data set excluding TCGA (non-TCGA ExAC) and the noncancer Genome Aggregation Database (gnomAD) as representative of population controls without a reported cancer diagnosis. We established both in vitro and murine in vivo models to functionally characterize representative variants.

Results: The existence of germline variants was first established in a family with wild-type who had oligopolyposis and early-onset colon cancers. A validation series indicated that germline variants occurred in 5 of 126 unrelated patients (4%) with oligopolyposis as a predominant phenotype. Germline variants, particularly the K740N and N745S alleles, were enriched in patients who did not have PHTS but had prevalent sporadic cancers, including related cancer types in TCGA (odds ratio, 1.5; 95% confidence interval, 1.1 to 2.1; P = 0.01). The prioritized variants resulted in gain-of-function effects, which led to aberrant enzymatic activation with consequent PTEN inactivation, thereby triggering hyperactive growth-promoting PI3K signaling in cellular and murine models.

Conclusions: In this study involving patients with disorders resulting in a predisposition to the development of multiple malignant neoplasms without germline mutations, we confirmed the function of as a cancer-susceptibility gene through direct aberrant regulation of the PTEN-PI3K signaling axis. (Funded by the National Institutes of Health and others.).
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http://dx.doi.org/10.1056/NEJMoa1914919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839065PMC
May 2020

Detection of circulating colorectal cancer cells by a custom microfluid system before and after endoscopic metallic stent placement.

Oncol Lett 2019 Dec 4;18(6):6397-6404. Epub 2019 Nov 4.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Although the detection of circulating tumor cells (CTCs) should be crucial for future personalized medicine, no efficient and flexible methods have been established. The current study established a polymeric custom-made chip for capturing CTCs with a high efficiency and flexibility. As an example of clinical application, the effects of self-expandable metallic stent (SEMS) placement on the release of cancer cells into the blood of patients with colorectal cancer and bowel obstruction were analyzed. This was assessed as the placement of SEMS may cause mechanical damage and physical force to malignant tissue, increasing the risk of cancer cell release into the bloodstream. The present study examined the number of CTCs using a custom-made chip, before, at 24 h after and at 4 days after SEMS placement in patients with colorectal cancer. The results revealed that, among the 13 patients examined, the number of CTCs was increased in three cases at 24 h after SEMS placement. However, this increase was temporary. The number of CTCs also decreased at 4 days after stent placement in most cases. The CTC chip of the current study detected the number of CD133-positive cancer stem-like cells, which did not change, even in the patient whose total number of CTCs temporarily increased. The results indicated that this custom-made microfluid system can efficiently and flexibly detect CTCs, demonstrating its potential for obtaining information during the management of patients with cancer.
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http://dx.doi.org/10.3892/ol.2019.11047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876337PMC
December 2019

Expression of circular RNA CDR1‑AS in colon cancer cells increases cell surface PD‑L1 protein levels.

Oncol Rep 2019 Oct 19;42(4):1459-1466. Epub 2019 Jul 19.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113‑8655, Japan.

The expression of CDR1‑AS, a representative circular RNA, is closely linked with poor prognosis in gastrointestinal cancers, such as colon, liver, and pancreatic cancers. Although it is well known that CDR1‑AS antagonizes microRNA‑7 function through its sequence similarities in the brain, its biological function and link with the malignant potential of cancer cells remain unclear, partly due to the difficulties of ectopic expression of circular RNAs. In the present study, SW620, a colon cancer cell line that stably expresses CDR1‑AS RNA circularized, was established using the laccase 2 gene cassette, and its biological function associated with malignant behavior was determined. In contrast to previous studies, cell growth or invasion ability was not altered by CDR1‑AS expression. However, the expression levels of CMTM4 and CMTM6, which were recently recognized as critical regulators of PD‑L1 protein expression at the cell surface, were significantly increased. Accordingly, the cell surface PD‑L1 protein levels were increased in CDR1‑AS‑expressing cells. Notably, the effects were not canceled out by overexpressing microRNA‑7, indicating that the increase in cell surface PD‑L1 in CDR1‑AS‑expressing cells was not dependent on microRNA‑7 function. These results indicated that expression of this circular RNA in cancer cells may lead to poor prognosis by increasing cell surface PD‑L1 levels through microRNA‑7‑independent mechanisms.
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http://dx.doi.org/10.3892/or.2019.7244DOI Listing
October 2019

Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx-DDB1 Interaction.

Cell Mol Gastroenterol Hepatol 2019 24;7(2):297-312. Epub 2018 Oct 24.

Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Background & Aims: Hepatitis B virus (HBV) infection is a major health concern worldwide. Although currently used nucleos(t)ide analogs efficiently inhibit viral replication, viral proteins transcribed from the episomal viral covalently closed circular DNA (cccDNA) minichromosome continue to be expressed long-term. Because high viral RNA or antigen loads may play a biological role during this chronicity, the elimination of viral products is an ultimate goal of HBV treatment. HBV regulatory protein X (HBx) was recently found to promote transcription of cccDNA with degradation of Smc5/6 through the interaction of HBx with the host protein DDB1. Here, this protein-protein interaction was considered as a new molecular target of HBV treatment.

Methods: To identify candidate compounds that target the HBx-DDB1 interaction, a newly constructed split luciferase assay system was applied to comprehensive compound screening. The effects of the identified compounds on HBV transcription and cccDNA maintenance were determined using HBV minicircle DNA, which mimics HBV cccDNA, and the natural HBV infection model of human primary hepatocytes.

Results: We show that nitazoxanide (NTZ), a thiazolide anti-infective agent that has been approved by the FDA for protozoan enteritis, efficiently inhibits the HBx-DDB1 protein interaction. NTZ significantly restores Smc5 protein levels and suppresses viral transcription and viral protein production in the HBV minicircle system and in human primary hepatocytes naturally infected with HBV.

Conclusions: These results indicate that NTZ, which targets an HBV-related viral-host protein interaction, may be a promising new therapeutic agent and a step toward a functional HBV cure.
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http://dx.doi.org/10.1016/j.jcmgh.2018.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357790PMC
May 2019

Pevonedistat, a Neuronal Precursor Cell-Expressed Developmentally Down-Regulated Protein 8-Activating Enzyme Inhibitor, Is a Potent Inhibitor of Hepatitis B Virus.

Hepatology 2019 05 13;69(5):1903-1915. Epub 2019 Mar 13.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan.

Hepatitis B virus (HBV) infection is a major health concern worldwide. To prevent HBV-related mortality, elimination of viral proteins is considered the ultimate goal of HBV treatment; however, currently available nucleos(t)ide analogs rarely achieve this goal, as viral transcription from episomal viral covalently closed circular DNA (cccDNA) is not prevented. HBV regulatory protein X was recently found to target the protein structural maintenance of chromosomes 5/6 (Smc5/6) for ubiquitination and degradation by DDB1-CUL4-ROC1 E3 ligase, resulting in enhanced viral transcription from cccDNA. This ubiquitin-dependent proteasomal pathway requires an additional ubiquitin-like protein for activation, neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8). Here, we show that pevonedistat, a NEDD8-activating enzyme inhibitor, works efficiently as an antiviral agent. Pevonedistat significantly restored Smc5/6 protein levels and suppressed viral transcription and protein production in the HBV minicircle system in in vitro HBV replication models and in human primary hepatocytes infected naturally with HBV. Conclusion: These results indicate that pevonedistat is a promising compound to treat chronic HBV infection.
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http://dx.doi.org/10.1002/hep.30491DOI Listing
May 2019

ISGF3 with reduced phosphorylation is associated with constitutive expression of interferon-induced genes in aging cells.

NPJ Aging Mech Dis 2018 15;4:11. Epub 2018 Nov 15.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655 Japan.

During cellular aging, many changes in cellular functions occur. A hallmark of aged cells is secretion of inflammatory mediators, which collectively is referred to as the senescence-associated secretory phenotype (SASP). However, the mechanisms underlying such changes are unclear. Canonically, the expression of interferon (IFN)-stimulated genes (ISGs) is induced by IFNs through the formation of the tripartite transcriptional factor ISGF3, which is composed of IRF9 and the phosphorylated forms of STAT1 and STAT2. However, in this study, the constitutive expression of ISGs in human-derived senescent fibroblasts and in fibroblasts from a patient with Werner syndrome, which leads to premature aging, was mediated mainly by the unphosphorylated forms of STATs in the absence of INF production. Under homeostatic conditions, STAT1, STAT2, and IRF9 were localized to the nucleus of aged cells. Although knockdown of JAK1, a key kinase of STAT1 and STAT2, did not affect ISG expression or IFN-stimulated response element (ISRE)-mediated promoter activities in these senescent cells, knockdown of STAT1 or STAT2 decreased ISG expression and ISRE activities. These results suggest that the ISGF3 complex without clear phosphorylation is required for IFN-independent constitutive ISG transcription in senescent cells.
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http://dx.doi.org/10.1038/s41514-018-0030-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237867PMC
November 2018

A phase II trial of gemcitabine, S-1 and LV combination (GSL) therapy in patients with advanced pancreatic cancer.

Invest New Drugs 2019 04 9;37(2):338-344. Epub 2018 Nov 9.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo, 113-8655, Japan.

Purpose Our previous phase I trial suggested feasibility of addition of leucovorin (LV) to S-1 and gemcitabine therapy in advanced pancreatic cancer. The aim of this phase II trial was to assess the efficacy and toxicity of gemcitabine, S-1 and LV (GSL) combination therapy for advanced pancreatic cancer. Methods Chemotherapy-naïve patients with histologically or cytologically proven advanced pancreatic cancer were enrolled. Gemcitabine was administered at a dose of 1000 mg/m2 by 30 min infusion on days 1, S-1 40 mg/m2 orally twice daily and LV 25 mg orally twice daily on days 1 to 7 every 2 weeks. Primary end point was progression free survival (PFS). Results A total of 49 patients with advanced pancreatic cancer (19 locally advanced and 30 metastatic) were enrolled. Overall response rate and disease control rate were 32.7% and 87.8%. The median PFS and overall survival (OS) were 10.8 (95% confidence interval [CI], 7.4-13.5) and 20.7 (95% CI 13.0-NA) months with 1-year survival rate of 73.4%. Major Grade 3-4 toxicities were neutropenia (22.4%) and stomatitis (14.3%). No toxicity related death was observed. Conclusions In this single center, phase II trial, gemcitabine, S-1 and LV combination therapy was tolerable and can potentially be a treatment option for advanced pancreatic cancer.
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http://dx.doi.org/10.1007/s10637-018-0691-9DOI Listing
April 2019

Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA.

World J Gastroenterol 2018 Jun;24(21):2261-2268

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Hepatitis B virus (HBV) is still a worldwide health concern. While divergent factors are involved in its pathogenesis, it is now clear that HBV RNAs, principally templates for viral proteins and viral DNAs, have diverse biological functions involved in HBV pathogenesis. These functions include viral replication, hepatic fibrosis and hepatocarcinogenesis. Depending on the sequence similarities, HBV RNAs may act as sponges for host miRNAs and may deregulate miRNA functions, possibly leading to pathological consequences. Some parts of the HBV RNA molecule may function as viral-derived miRNA, which regulates viral replication. HBV DNA can integrate into the host genomic DNA and produce novel viral-host fusion RNA, which may have pathological functions. To date, elimination of HBV-derived covalently closed circular DNA has not been achieved. However, RNA transcription silencing may be an alternative practical approach to treat HBV-induced pathogenesis. A full understanding of HBV RNA transcription and the biological functions of HBV RNA may open a new avenue for the development of novel HBV therapeutics.
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http://dx.doi.org/10.3748/wjg.v24.i21.2261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989240PMC
June 2018

DHX9 regulates production of hepatitis B virus-derived circular RNA and viral protein levels.

Oncotarget 2018 Apr 20;9(30):20953-20964. Epub 2018 Apr 20.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Hepatitis B virus (HBV) infection, which is a major health concern worldwide, can lead to liver cirrhosis and hepatocellular carcinoma. Although current nucleos(t)ide analogs efficiently inhibit viral reverse transcription and viral DNA load clinically, episomal viral covalently closed circular DNA (cccDNA) minichromosomes and transcripts from cccDNA continue to be expressed over the long term. We hypothesized that, under these conditions, viral transcripts may have biological functions involved in pathogenesis. Here, we show that the host protein DExH-box helicase 9 (DXH9) is associated with viral RNAs. We also show that viral-derived circular RNA is produced during HBV replication, and the amount is increased by knockdown of the DHX9 protein, which, in turn, results in decreased viral protein levels but does not affect the levels of HBV DNA. These phenomena were observed in the HBV-producing cell culture model and HBV mini-circle model mimicking HBV cccDNA, as well as in human primary hepatocytes infected with HBV. Based on these results, we conclude that, in HBV infection, the RNA binding factor DHX9 is a novel regulator of viral circular RNA and viral protein levels.
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http://dx.doi.org/10.18632/oncotarget.25104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940377PMC
April 2018

Satellite RNA Increases DNA Damage and Accelerates Tumor Formation in Mouse Models of Pancreatic Cancer.

Mol Cancer Res 2018 08 10;16(8):1255-1262. Epub 2018 May 10.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Highly repetitive tandem arrays such as satellite sequences in the centromeric and pericentromeric regions of chromosomes, which were previously considered to be silent, are actively transcribed in various biological processes, including cancers. In the pancreas, this aberrant expression occurs even in Kras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are precancerous lesions. To determine the biological role of satellite RNAs in carcinogenesis , we constructed mouse major satellite (MajSAT) RNA-expressing transgenic mice. However, these transgenic mice did not show spontaneous malignant tumor formation under normal breeding. Importantly, however, DNA damage was increased in pancreatic tissues induced by caerulein treatment or high-fat diet, which may be due to impaired nuclear localization of Y-Box Binding Protein 1 (YBX1), a component of the DNA damage repair machinery. In addition, when crossed with pancreas-specific Kras-mutant mice, MajSAT RNA expression resulted in an earlier increase in PanIN formation. These results suggest that aberrant MajSAT RNA expression accelerates oncogenesis by increasing the probability of a second driver mutation, thus accelerating cells to exit from the breakthrough phase to the expansion phase. Aberrant expression of satellite RNAs accelerates oncogenesis through a mechanism involving increased DNA damage. .
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0139DOI Listing
August 2018

RASAL1 is a potent regulator of hepatic stellate cell activity and liver fibrosis.

Oncotarget 2017 Sep 4;8(39):64840-64852. Epub 2017 May 4.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Liver fibrosis, leading to cirrhosis and liver failure, can occur after chronic liver injury. The transition of hepatic stellate cells (HSCs) from quiescent cells into proliferative and fibrogenic cells is a central event in liver fibrosis. Here, we show that RAS protein activator like-1 (RASAL1), a RAS-GTPase-activating protein, which switches off RAS activity, is significantly decreased during HSC activation, and that HSC activation can be antagonized by forced expression of the RASAL1 protein. We demonstrate that RASAL1 suppresses HSC proliferation by regulating the Ras-MAPK pathway, and that RASAL1 suppresses HSC fibrogenic activity by regulating the PKA-LKB1-AMPK-SRF pathway by interacting with angiotensin II receptor, type 1. We also show that RASAL1-deficient mice are more susceptible to liver fibrosis. These data demonstrate that deregulated RASAL1 expression levels and the affected downstream intracellular signaling are central mediators of perpetuated HSC activation and fibrogenesis in the liver.
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http://dx.doi.org/10.18632/oncotarget.17609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630295PMC
September 2017

Transcriptional activation of the MICA gene with an engineered CRISPR-Cas9 system.

Biochem Biophys Res Commun 2017 04 18;486(2):521-525. Epub 2017 Mar 18.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Major histocompatibility complex class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. Because immune cells, such as natural killer (NK) cells, recognize virally infected or transformed cells and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells, MICA expression levels are associated with NK cell-mediated immunity. Here, we report that an engineered clustered regularly interspaced short palindromic repeats-Cas9-related complex targeting MICA gene promoter sequences activates transcription of the MICA gene from its endogenous locus. Inhibiting microRNA function, which targets the 3' untranslated region of the MICA gene, enhances this activation. These results demonstrate that the combination of Cas9-based transcriptional activators and simultaneous modulation of microRNA function may be a powerful tool for enhancing MICA protein expression and efficient anti-pathogenic cell immunity.
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http://dx.doi.org/10.1016/j.bbrc.2017.03.076DOI Listing
April 2017

Repression of MicroRNA Function Mediates Inflammation-associated Colon Tumorigenesis.

Gastroenterology 2017 02 5;152(3):631-643. Epub 2016 Nov 5.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Background & Aims: Little is known about the mechanisms by which chronic inflammation contributes to carcinogenesis, such as the development of colon tumors in patients with inflammatory bowel diseases. Specific microRNA (miRNAs) can function as suppressors or oncogenes, and widespread alterations in miRNA expression have been associated with tumorigenesis. We studied whether alterations in miRNA function contribute to inflammation-associated colon carcinogenesis.

Methods: We studied the effects of inflammatory cytokines, such as tumor necrosis factor, interleukin-1α (IL1A), and IL1β (IL1B), on miRNA function, measured by activity of reporter constructs containing miRNA-binding sites in their 3' untranslated regions, in human 293T embryonic kidney, Caco-2, HT29, and HCT116 colon carcinoma cells, as well as dicer and dicer, and Apobec3 and Apobec3 mouse embryonic fibroblasts. Cells were analyzed by immunoblots, immunohistochemistry, and flow cytometry. We generated transgenic mice expressing reporter constructs regulated by LET7B, MIR122, and MIR29b response elements; some mice were given injections of miRNA inhibitors (anti-MIR122 or anti-LET7B), a negative control, or tumor necrosis factor. Liver tissues were collected and analyzed by immunoblotting. Reporter mice were given azoxymethane followed by dextran sulfate sodium to induce colitis and colon tumors; some mice were given the ROCK inhibitor fasudil along with these agents (ROCK inhibitors increase miRNA function). Colon tissues were collected and analyzed by immunohistochemistry, immunoblots, and fluorescence microscopy.

Results: Incubation of cell lines with inflammatory cytokines reduced the ability of miRNAs to down-regulate expression from reporter constructs; dicer was required for this effect, so these cytokines relieve miRNA-dependent reductions in expression. The cytokines promoted degradation of APOBEC3G, which normally promotes miRNA loading into argonaute 2-related complexes. Mice with colitis had reduced miRNA function, based on increased expression of reporter genes. Administration of fasudil to mice did not reduce the severity of colitis that developed but greatly reduced the numbers of colon tumors formed (mean 2 tumors/colon in mice given fasudil vs 9 tumors/colon in mice given control agent). We made similar observations in IL10-deficient mice.

Conclusions: We found inflammatory cytokines to reduce the activities of miRNAs. In mice with colitis, activities of miRNAs are reduced; administration of an agent that increases miRNA function prevents colon tumor formation in these mice. This pathway might be targeted to prevent colon carcinogenesis in patients with inflammatory bowel diseases.
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http://dx.doi.org/10.1053/j.gastro.2016.10.043DOI Listing
February 2017

Quantitation of circulating satellite RNAs in pancreatic cancer patients.

JCI Insight 2016 06 2;1(8):e86646. Epub 2016 Jun 2.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Pancreatic ductal adenocarcinoma (Pdac) is a malignancy with a poor prognosis due to difficulties in early detection. Although promising biomarkers are increasingly reported, such methods are not yet easy to apply clinically, mainly due to their low reproducibility or technical difficulties. In this study, we developed a convenient and sensitive method for quantifying aberrantly expressed satellite repeat RNAs in sera, which can be used to efficiently detect patients with Pdac. Here, we introduce a Tandem Repeat Amplification by nuclease Protection (TRAP) method combined with droplet digital PCR (ddPCR) to detect human satellite II (HSATII) RNAs, which are specifically expressed in human Pdacs at greater levels than normal tissues but are difficult to measure due to their repetitive sequences and irregularities. HSATII RNA core sequence levels in sera were significantly higher in Pdac patients compared with noncancer patients (median copy number: 14.75 and 3.17 per μl in the training set and 17.35 and 2.9 in the validation set, respectively). In addition, patients with intraductal papillary mucinous neoplasm (IPMN), a precancerous lesion of Pdac, could also be efficiently detected. This method can be routinely applied to screen patients with Pdac and high-risk patients, facilitating the development of preventive medicine for this disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033942PMC
http://dx.doi.org/10.1172/jci.insight.86646DOI Listing
June 2016

Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1.

Nat Commun 2016 Sep 26;7:13006. Epub 2016 Sep 26.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Highly repetitive tandem arrays at the centromeric and pericentromeric regions in chromosomes, previously considered silent, are actively transcribed, particularly in cancer. This aberrant expression occurs even in K-ras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are precancerous lesions. To examine the biological roles of the satellite RNAs in carcinogenesis, we construct mouse PanIN-derived cells expressing major satellite (MajSAT) RNA and show increased malignant properties. We find an increase in frequency of chromosomal instability and point mutations in both genomic and mitochondrial DNA. We identify Y-box binding protein 1 (YBX1) as a protein that binds to MajSAT RNA. MajSAT RNA inhibits the nuclear translocation of YBX1 under stress conditions, thus reducing its DNA-damage repair function. The forced expression of YBX1 significantly decreases the aberrant phenotypes. These findings indicate that during the early stage of cancer development, satellite transcripts may act as 'intrinsic mutagens' by inducing YBX1 dysfunction, which may be crucial in oncogenic processes.
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http://dx.doi.org/10.1038/ncomms13006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052683PMC
September 2016

MicroRNAs and liver disease.

J Hum Genet 2017 Jan 26;62(1):75-80. Epub 2016 May 26.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

The biological roles of microRNAs (miRNAs) have been extensively studied. miRNA122 represents more than half of the miRNAs expressed in the liver and has various physiological and pathological functions, which include enhancing hepatitis virus replication, regulating lipid metabolism and suppressing hepatocellular carcinoma. miRNAs, whether globally or individually, have been linked with hepatocarcinogenesis. Furthermore, some miRNAs have been shown to be involved in the pathogenesis of nonalcoholic steatohepatitis. Using nucleotide-based strategies, these miRNAs may be developed as potential therapeutic targets. Because changes in miRNA expression can be measured in sera, they may be used as non-invasive biomarkers if they correctly reflect the pathological state of the liver. In this review, we show the biological roles of representative miRNAs in liver disease and discuss the current issues that remain to be clarified for future clinical applications.
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http://dx.doi.org/10.1038/jhg.2016.53DOI Listing
January 2017

Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7.

Sci Rep 2016 Mar 16;6:23237. Epub 2016 Mar 16.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection.
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http://dx.doi.org/10.1038/srep23237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4793232PMC
March 2016

Development of a screening method to identify regulators of MICA shedding.

Biochem Biophys Res Commun 2015 Oct 20;465(4):764-8. Epub 2015 Aug 20.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Immune cells, such as natural killer (NK) cells, recognize virally infected and transformed cells, and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells. Shedding of NKG2D ligands is thought to be a type of counter-mechanism employed by pathogenic cells to evade from NKG2D-mediated immune surveillance. MHC class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. We previously reported that, in soluble form, MICA expression levels are significantly associated with hepatitis virus-induced hepatocellular carcinoma. Here, we report a MICA shedding assay that utilizes membrane-bound MICA tagged at its N-terminus with a nano-luciferase reporter to quantify MICA shedding into culture media. Using this method, we screened a compound library and identified putative regulators of MICA shedding that have the potential to enhance the immune reaction by simultaneously increasing cell surface MICA levels and decreasing soluble MICA levels. This shedding assay may be useful for screening regulators of cell surface molecule shedding.
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http://dx.doi.org/10.1016/j.bbrc.2015.08.081DOI Listing
October 2015

Circulating RNAs as new biomarkers for detecting pancreatic cancer.

World J Gastroenterol 2015 Jul;21(28):8527-40

Takahiro Kishikawa, Motoyuki Otsuka, Motoko Ohno, Takeshi Yoshikawa, Akemi Takata, Kazuhiko Koike, Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Pancreatic cancer remains difficult to treat and has a high mortality rate. It is difficult to diagnose early, mainly due to the lack of screening imaging modalities and specific biomarkers. Consequently, it is important to develop biomarkers that enable the detection of early stage tumors. Emerging evidence is accumulating that tumor cells release substantial amounts of RNA into the bloodstream that strongly resist RNases in the blood and are present at sufficient levels for quantitative analyses. These circulating RNAs are upregulated in the serum and plasma of cancer patients, including those with pancreatic cancer, compared with healthy controls. The majority of RNA biomarker studies have assessed circulating microRNAs (miRs), which are often tissue-specific. There are few reports of the tumor-specific upregulation of other types of small non-coding RNAs (ncRNAs), such as small nucleolar RNAs and Piwi-interacting RNAs. Long ncRNAs (lncRNAs), such as HOTAIR and MALAT1, in the serum/plasma of pancreatic cancer patients have also been reported as diagnostic and prognostic markers. Among tissue-derived RNAs, some miRs show increased expression even in pre-cancerous tissues, and their expression profiles may allow for the discrimination between a chronic inflammatory state and carcinoma. Additionally, some miRs and lncRNAs have been reported with significant alterations in expression according to disease progression, and they may thus represent potential candidate diagnostic or prognostic biomarkers that may be used to evaluate patients once detection methods in peripheral blood are well established. Furthermore, recent innovations in high-throughput sequencing techniques have enabled the discovery of unannotated tumor-associated ncRNAs and tumor-specific alternative splicing as novel and specific biomarkers of cancers. Although much work is required to clarify the release mechanism, origin of tumor-specific circulating RNAs, and selectivity of carrier complexes, and technical advances must also be achieved, such as creating a consensus normalization protocol for quantitative data analysis, circulating RNAs are largely unexplored and might represent novel clinical biomarkers.
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http://dx.doi.org/10.3748/wjg.v21.i28.8527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515835PMC
July 2015

ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

Nucleic Acids Res 2015 Sep 17;43(15):7577-89. Epub 2015 Jul 17.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.
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http://dx.doi.org/10.1093/nar/gkv728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551943PMC
September 2015

Novel therapeutic approaches for hepatitis B virus covalently closed circular DNA.

World J Gastroenterol 2015 Jun;21(23):7084-8

Motoko Ohno, Motoyuki Otsuka, Takahiro Kishikawa, Takeshi Yoshikawa, Akemi Takata, Kazuhiko Koike, Department of Gastroenterology, Graduate School of Medicine, the University of Tokyo, Tokyo 113-8655, Japan.

Hepatitis B virus (HBV) infection is a major global health problem. Although current therapies, such as the use of nucleos(t)ide analogs, inhibit HBV replication efficiently, they do not eliminate covalently closed circular DNA (cccDNA), which persists in hepatocyte nuclei. As HBV cccDNA is a viral transcription template, novel therapeutic approaches to directly target HBV cccDNA are necessary to completely eradicate persistent HBV infections. HBV cccDNA levels in HBV-infected human liver cells are extremely low; thus, more reliable and simple measurement methods are needed to correctly monitor their levels during therapeutic treatment. Although reverse transcription-polymerase chain reaction or Southern blot procedures are currently used in research studies, these methods are not completely reliable and are also time-consuming and labor-intensive. Genome editing technologies, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which are designed to target specific DNA sequences, represent highly promising potential therapeutic tools. In particular, the CRISPR/Cas9 system is an easily customizable sequence-specific nuclease with high flexibility and may be the most feasible approach to target HBV cccDNA. Further research to develop easier, safer, and more effective protocols should be pursued.
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http://dx.doi.org/10.3748/wjg.v21.i23.7084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476870PMC
June 2015

Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine.

Oncotarget 2015 Apr;6(10):8339-52

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480756PMC
http://dx.doi.org/10.18632/oncotarget.3234DOI Listing
April 2015

Diagnostic and therapeutic application of noncoding RNAs for hepatocellular carcinoma.

World J Hepatol 2015 Jan;7(1):1-6

Chikako Shibata, Motoyuki Otsuka, Takahiro Kishikawa, Motoko Ohno, Takeshi Yoshikawa, Akemi Takata, Kazuhiko Koike, Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression posttranscriptionally, targeting thousands of messenger RNAs. Long noncoding RNAs (lncRNAs), another class of noncoding RNAs, have been determined to be also involved in transcription regulation and translation of target genes. Since deregulated expression levels or functions of miRNAs and lncRNAs in hepatocellular carcinoma (HCC) are frequently observed, clinical use of noncoding RNAs for novel diagnostic and therapeutic applications in the management of HCCs is highly and emergently expected. Here, we summarize recent findings regarding deregulated miRNAs and lncRNAs for their potential clinical use as diagnostic and prognostic biomarkers of HCC. Specifically, we emphasize the deregulated expression levels of such noncoding RNAs in patients' sera as noninvasive biomarkers, a field that requires urgent improvement in the clinical surveillance of HCC. Since nucleotide-based strategies are being applied to clinical therapeutics, we further summarize clinical and preclinical trials using oligonucleotides involving the use of miRNAs and small interfering RNAs against HCC as novel therapeutics. Finally, we discuss current open questions, which must be clarified in the near future for realistic clinical applications of these new strategies.
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http://dx.doi.org/10.4254/wjh.v7.i1.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295186PMC
January 2015

The flavonoid apigenin inhibits hepatitis C virus replication by decreasing mature microRNA122 levels.

Virology 2014 Aug 14;462-463:42-8. Epub 2014 Jun 14.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 5-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

Despite recent progress in the development of direct-acting antivirals against hepatitis C virus (HCV), chronic HCV infection remains an important health burden worldwide. MicroRNA122 (miR122), a liver-specific microRNA (miRNA), positively regulates HCV replication, and systemic application of antisense oligonucleotides against miR122 led to the long-lasting suppression of HCV viremia in human clinical trials. Here, we report that apigenin, a flavonoid and an inhibitor of maturation of a subset of miRNAs, inhibits HCV replication in vitro. Apigenin decreased the expression levels of mature miR122 without significantly affecting cell growth. Because supplementation of synthesized miR122 oligonucleotides or overexpression of constitutively active TRBP blocked these effects, the inhibitory effects of apigenin on HCV replication seemed to be dependent on the reduction of mature miR122 expression levels through inhibition of TRBP phosphorylation. Thus, apigenin intake, either through regular diet or supplements, may decrease HCV replication in chronically infected patients.
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http://dx.doi.org/10.1016/j.virol.2014.05.024DOI Listing
August 2014

Specific delivery of microRNA93 into HBV-replicating hepatocytes downregulates protein expression of liver cancer susceptible gene MICA.

Oncotarget 2014 Jul;5(14):5581-90

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). To date, the lack of efficient in vitro systems supporting HBV infection and replication has been a major limitation of HBV research. Although primary human hepatocytes support the complete HBV life cycle, their limited availability and difficulties with gene transduction remain problematic. Here, we used human primary hepatocytes isolated from humanized chimeric uPA/SCID mice as efficient sources. These hepatocytes supported HBV replication in vitro. Based on analyses of mRNA and microRNA (miRNA) expression levels in HBV-infected hepatocytes, miRNA93 was significantly downregulated during HBV infection. MiRNA93 is critical for regulating the expression levels of MICA protein, which is a determinant for HBV-induced HCC susceptibility. Exogenous addition of miRNA93 in HBV-infected hepatocytes using bionanocapsules consisted of HBV envelope L proteins restored MICA protein expression levels in the supernatant. These results suggest that the rescued suppression of soluble MICA protein levels by miRNA93 targeted to HBV-infected hepatocytes using bionanocapsules may be useful for the prevention of HBV-induced HCC by altering deregulated miRNA93 expression.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4170619PMC
http://dx.doi.org/10.18632/oncotarget.2143DOI Listing
July 2014

The role of microRNAs in hepatocarcinogenesis: current knowledge and future prospects.

J Gastroenterol 2014 Feb 21;49(2):173-84. Epub 2013 Nov 21.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 5-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan,

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression post-transcriptionally through complementary base pairing with thousands of messenger RNAs. Although the precise biological functions of individual miRNAs are still unknown, miRNAs are speculated to play important roles in diverse biological processes through fine regulation of their target gene expression. A growing body of data indicates the deregulation of miRNAs during hepatocarcinogenesis. In this review, we summarize recent findings regarding deregulated miRNA expression and their possible target genes in hepatocarcinogenesis, with emphasis on inflammation-related hepatocarcinogenesis. Because miRNA-based strategies are being applied to clinical therapeutics, precise knowledge of miRNA functions is crucial both scientifically and clinically. We discuss the current open questions from these points of view, which must be clarified in the near future.
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http://dx.doi.org/10.1007/s00535-013-0909-8DOI Listing
February 2014

Regulation of the expression of the liver cancer susceptibility gene MICA by microRNAs.

Sci Rep 2013 ;3:2739

1] Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan [2].

Hepatocellular carcinoma (HCC) is a threat to public health worldwide. We previously identified the association of a single nucleotide polymorphism (SNP) at the promoter region of the MHC class I polypeptide-related sequence A (MICA) gene with the risk of hepatitis-virus-related HCC. Because this SNP affects MICA expression levels, regulating MICA expression levels may be important in the prevention of HCC. We herein show that the microRNA (miR) 25-93-106b cluster can modulate MICA levels in HCC cells. Overexpression of the miR 25-93-106b cluster significantly suppressed MICA expression. Conversely, silencing of this miR cluster enhanced MICA expression in cells that express substantial amounts of MICA. The changes in MICA expression levels by the miR25-93-106b cluster were biologically significant in an NKG2D-binding assay and an in vivo cell-killing model. These data suggest that the modulation of MICA expression levels by miRNAs may be a useful method to regulate HCCs during hepatitis viral infection.
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http://dx.doi.org/10.1038/srep02739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781398PMC
July 2014
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