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Chromatin Immunoprecipitation and DNA Sequencing Identified A CANCER GENOMICS & PROTEOMICS 15 : 165-174 (2018) doi:10.21873/cgp.20074 Chromatin Immunoprecipitation and DNA Sequencing Identified a LIMS1/ILK Pathway Regulated by LMO1 in Neuroblastoma NORIHISA SAEKI 1* , AKIRA SAITO 2, YUKI SUGAYA 2, MITSUHIRO AMEMIYA 2, HIROE ONO 1, RIE KOMATSUZAKI 3, KAZUYOSHI YANAGIHARA 4 and HIROKI SASAKI 3 1Division of Genetics, National Cancer Center Research Institute, Tokyo, Japan; 2Statistical Genetics Analysis Division, StaGen Co. Ltd., Tokyo, Japan; 3Department of Translational Oncology, National Cancer Center Research Institute, Tokyo, Japan; 4Division of Pathology, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center Hospital East, Chiba, Japan Abstract. Background/Aim: Overall survival for the high- malignancies in patients younger than 15, it is a cause for risk group of neuroblastoma (NB) remains at 40-50%. An approximately 15% of all pediatric cancer deaths (1). NB integrative genomics study revealed that LIM domain only 1 cells develop from a sympathicoadrenal linage of the neural (LMO1) encoding a transcriptional regulator to be an NB- crest and form solid tumors in the adrenal medulla or susceptibility gene with a tumor-promoting activity, that paraspinal regions, which cause variable symptoms by needs to be revealed. Materials and Methods: We conducted compressing and/or invading to adjacent organs depending chromatin immunoprecipitation and DNA sequencing on their location (1-3). NB cells are heterogenous, and analyses and cell proliferation assays on two NB cell lines. patient age at the time of diagnosis is one of the major Results: We identified three genes regulated by LMO1 in the factors that contribute to the determination of their biological cells, LIM and senescent cell antigen-like domains 1 characters; patients older than 18 months are considered to (LIMS1), Ras suppressor protein 1 (RSU1) and relaxin 2 have a poorer prognosis when associated with dissemination (RLN2). LIMS1 and RSU1 encode proteins functioning with than are patients younger than 18 months (1). In the context integrin-linked kinase (ILK), and inhibition of LIMS1, ILK of prognosis, patients with NB are categorized into 4 groups: or RLN2 by shRNA reduced cell proliferation of the NB cells, very low risk, low risk (LR), intermediate risk (IR) and high which was also suppressed with an ILK inhibiting compound risk (HR), with a 5-year event-free survival of >85%, >75 to Cpd 22. Conclusion: The downstream of LMO1-regulatory ≤85%, ≥50 to ≤75%, and <50%, respectively, based on age cascade includes a tumor-promoting LIMS1/ILK pathway, at diagnosis, International Neuroblastoma Risk Group which has a potential to be a novel therapeutic target. (INRG) tumour stage, histologic category, grade of tumor differentiation, DNA ploidy, and copy-number status at the NB is the most common extracranial solid tumor in MYCN oncogene locus and at chromosome 11q (1). Recent childhood. Although accounting for more than 7% of progress in NB treatment significantly improved patient prognosis; overall survival (OS) for LR and IR groups is now >98% and 90 to 95%, respectively. However, OS for the This article is freely accessible online. HR group still remains at 40 to 50%, and 50 to 60% of these patients have a relapse, which, in that case, leaves no option *Present address: Division of Anatomy and Physiology, Okinawa of curable treatment (1-5). To improve the OS and quality of Prefectural College of Nursing, Okinawa, Japan life for the HR group, the first and mandatory line of investigation is the identification of novel therapeutic target Correspondence to: Norihisa Saeki, Division of Anatomy and molecules in NB cells (6-8). Physiology, Okinawa Prefectural College of Nursing, Yogi 1-24-1, Currently, a number of genome-wide association studies Naha, Okinawa 902-0076, Japan. Tel: +81 988338885, Fax: +81 988335133, e-mail: [email protected] (GWASs) have been performed for identifying disease susceptibility-associated genes using disease-related genetic Key Words: Neuroblastoma, pediatric cancer, LMO1, ChIP-seq, variation, and the LIM domain only 1 ( LMO1 ) gene was therapeutic targets, transcription regulator. identified as an NB susceptibility gene by a GWAS (9, 10). 165 CANCER GENOMICS & PROTEOMICS 15 : 165-174 (2018) The study also revealed that both the presence of an NB- Developed TaqMan Assay Reagent for glyceraldehyde-3-phosphate related allele of single nucleotide polymorphism (SNP) dehydrogenase ( GAPDH )). The PCR was performed for 40 cycles rs110419 in the LMO1 gene and the increased copy number under a condition of 2 steps of temperature: 95˚C for 15sec and 60˚C for 60sec, using ABI PRISM 7900HT Sequence Detection of the gene contribute to augmented LMO1 expression, and System (Life Technologies). The relative transcript level was that LMO1 has a role in the proliferation of NB cells. calculated using the Ct value of GAPDH transcript as reference. The Notably, the relation of the variation at rs110419 in LMO1 RNA was extracted from shRNA-introduced cells after 7-10 days’ as well as the increased copy number to NB is stronger in duration of G418 selection (Geneticin, WAKO). Microarray the HR group than in the non-HR. Thereafter, other studies expression analyses were performed with GeneChip Human also revealed the correlation between the SNP and NB Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA). susceptibility in different ethnic populations (11, 12). In addition, it was recently reported LMO1 has synergetic ChIP-seq. Chromatin samples of the NB cells were prepared using ChIP-IT Express Enzymatic Kit (Active Motif Japan, Tokyo, Japan), effects with MYCN on NB metastasis (13). Therefore, it is which were immunoprecipitated with the anti-LMO1 antibody and highly likely that, as a transcription regulator, LMO1 is Protein G sepharose (GE Healthcare Japan). DNA samples purified involved in the regulation of expression of variable genes from the precipitated chromatin were applied to the library important for NB progression in HR patients. Consequently, construction using ChIP-Seq Sample Prep Kit (Illumina K.K., Tokyo, it is anticipated that identification of the LMO1’s regulatory Japan) and Multiplexing Sample Preparation Oligonucleotide Kit targets may uncover novel tumor-promoting molecular (Illumina). In brief, after adapter ligation, around 300-bp fraction of pathways in NB, in which novel therapeutic target DNA was isolated by electrophoresis through agarose gel, and clusters for sequencing were prepared using TruSeq PE Cluster Kit molecules, especially for treatment of the HR group, will be v3-cBot-HS (Illumina). DNA sequencing was conducted by Takara discovered. Bio Inc. (Shiga, Japan) with HiSeq 2000 (Illumina) for 100 nucleotides in a single-read manner. Input DNA samples were also Materials and Methods sequenced as reference for data analyses. Cell lines. An NB cell line SK-N-SH (SH) was provided from Sequence data analyses . Quality check of the data was performed European Collection of Cell Cultures and maintained in D-MEM with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/ (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented fastqc/) which revealed a high quality of the sequence data (reads) with 10% fetal calf serum (FCS). LA-N-5 (LAN5) was provided by and all reads were applied to alignment of the human genome. Raw the RIKEN Bioresource Center through the National Bio-Resource sequencing reads were aligned to the UCSC hg19 reference genome Project of the MEXT, Japan and maintained in RPMI1640 (Life using Burrows-Wheeler Aligner (15) supplemented with Novoalign Technologies, Tokyo, Japan) supplemented with 10% FCS. (Novocraft Technologies, Selangor, Malaysia). As a result of the genome-alignment, 59,446,992 and 50,555,592 reads from the ChIP Immune precipitation and western blot analyses. A 3×FLAG-LMO1 sequence data of SH and LAN5, respectively, were selected for peak- expression vector was constructed by insertion of LMO1 cDNA (14) identification analyses, accompanied with their reference data. The into p3×FLAG-Myc-CMV (Sigma-Aldrich, St. Louis, MO USA). peaks of accumulation of the reads were detected using six programs: Cell lysates were prepared using CelLytic-M Mammalian Cell CisGenome, MACS versions one and two, SISSRs, QuEST, and SPP. Lysis/Extraction Reagent (Sigma-Aldrich) and Protease Inhibitor When using SISSRs, the p- value threshold was set to 0.1 and the Cocktail (Sigma-Aldrich). As preliminary experiment for selecting threshold of the number of expected false-positive positions ( e-value) anti-LMO1 antibodies, immune precipitation was conducted on cell was set to 500. Peak detection using QuEST was conducted with lysate of a gastric cancer cell line HSC-59 (14) introduced with a three different parameter settings: parameters for transcription factor 3×FLAG-LMO1 expression construct, using anti-FLAG (EZView (QuEST-TF), for polII-like factor (QuEST-PolII), and for histone-type Red ANTI-FLAG M2 Affinity Gel, Sigma-Aldrich) and anti-LMO1 ChIP (QuEST-His). Peak detection using SPP was performed by two antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). Immune methods: window tag density (WTD) and mirror tag correlation blotting was performed with the anti-FLAG (Sigma-Aldrich) or (MTC). The e-value was set to 1000 when using SPP (WTD and anti-LMO1 (Santa Cruz Biotechnology) antibody as primary MTC). The default settings were used for all other program options. antibodies and HRP-conjugated anti-mouse or anti-goat antibodies (Santa
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