ABSTRACT
BACKGROUND/AIM: Overall survival for the high-risk group of neuroblastoma (NB) patients still remains at 40-50%, necessitating the establishment of a curable treatment. LIM domain only 1 (LMO1) gene encoding a transcriptional regulator is an NB-susceptibility gene with a tumor-promoting activity. Previously we conducted chromatin immunoprecipitation and DNA sequencing analyses on NB cell lines and identified 3 protein-coding genes regulated by LMO1. In this study, we extended our analyses to capture microRNA genes directly or indirectly regulated by LMO1. MATERIALS AND METHODS: Using microarrays, we conducted a comparative gene expression analysis on an NB cell line SK-N-SH; between the cells with and without LMO1 suppression. RESULTS: Overall, 18 microRNAs were identified to be indirectly down-regulated by LMO1 including 7 microRNAs of the let-7 family, whose cell proliferation inhibitory activity was observed. CONCLUSION: Target genes of the LMO1-regulated microRNAs and their relevant pathways may be a potential therapeutic target.
Subject(s)
DNA-Binding Proteins/genetics , LIM Domain Proteins/genetics , MicroRNAs/genetics , Neuroblastoma/genetics , Transcription Factors/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation/genetics , Humans , Microarray Analysis/methods , Neuroblastoma/pathologyABSTRACT
BACKGROUND/AIM: Overall survival for the high-risk group of neuroblastoma (NB) remains at 40-50%. An integrative genomics study revealed that LIM domain only 1 (LMO1) encoding a transcriptional regulator to be an NB-susceptibility gene with a tumor-promoting activity, that needs to be revealed. MATERIALS AND METHODS: We conducted chromatin immunoprecipitation and DNA sequencing analyses and cell proliferation assays on two NB cell lines. RESULTS: We identified three genes regulated by LMO1 in the cells, LIM and senescent cell antigen-like domains 1 (LIMS1), Ras suppressor protein 1 (RSU1) and relaxin 2 (RLN2). LIMS1 and RSU1 encode proteins functioning with integrin-linked kinase (ILK), and inhibition of LIMS1, ILK or RLN2 by shRNA reduced cell proliferation of the NB cells, which was also suppressed with an ILK inhibiting compound Cpd 22. CONCLUSION: The downstream of LMO1-regulatory cascade includes a tumor-promoting LIMS1/ILK pathway, which has a potential to be a novel therapeutic target.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Chromatin Immunoprecipitation/methods , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , High-Throughput Nucleotide Sequencing/methods , LIM Domain Proteins/genetics , Neuroblastoma/genetics , Protein Serine-Threonine Kinases/genetics , Transcription Factors/genetics , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Cell Proliferation , DNA-Binding Proteins/antagonists & inhibitors , Humans , LIM Domain Proteins/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Neuroblastoma/metabolism , Neuroblastoma/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , RNA, Small Interfering/genetics , Signal Transduction , Transcription Factors/antagonists & inhibitors , Tumor Cells, CulturedABSTRACT
Next-generation sequencing (NGS) is widely used for the detection of disease-causing nucleotide variants. The challenges associated with detecting copy number variants (CNVs) using NGS analysis have been reported previously. Disease-related exome panels such as Illumina TruSight One are more cost-effective than whole-exome sequencing (WES) because of their selective target regions (~21% of the WES). In this study, CNVs were analyzed using data extracted through a disease-related exome panel analysis and the eXome Hidden Markov Model (XHMM). Samples from 61 patients with undiagnosed developmental delays and 52 healthy parents were included in this study. In the preliminary study to validate the constructed XHMM system (microarray-first approach), 34 patients who had previously been analyzed by chromosomal microarray testing were used. Among the five CNVs larger than 200 kb that were considered as non-pathogenic CNVs and were used as positive controls, four CNVs was successfully detected. The system was subsequently used to analyze different samples from 27 patients (NGS-first approach); 2 of these patients were successfully diagnosed as having pathogenic CNVs (an unbalanced translocation der(5)t(5;14) and a 16p11.2 duplication). These diagnoses were re-confirmed by chromosomal microarray testing and/or fluorescence in situ hybridization. The NGS-first approach generated no false-negative or false-positive results for pathogenic CNVs, indicating its high sensitivity and specificity in detecting pathogenic CNVs. The results of this study show the possible clinical utility of pathogenic CNV screening using disease-related exome panel analysis and XHMM.
