Dataset Information

Transcriptome stability profiling using 5'-bromouridine IP chase (BRIC-seq) identifies novel and functional microRNA targets in human melanoma cells.

ABSTRACT: RNA half-life is closely related to its cellular physiological function, so stability determinants may have regulatory functions. Micro(mi)RNAs have primarily been studied with respect to post-transcriptional mRNA regulation and target degradation. Here we study the impact of the tumour suppressive melanoma miRNA miR-211 on transcriptome stability and phenotype in the non-pigmented melanoma cell line, A375. Using 5'-bromouridine IP chase (BRIC)-seq, transcriptome-wide RNA stability profiles revealed highly regulated genes and pathways important in this melanoma cell line. By combining BRIC-seq, RNA-seq and in silico predictions, we identified both existing and novel direct miR-211 targets. We validated DUSP3 as one such novel miR-211 target, which itself sustains colony formation and invasion in A375 cells via MAPK/PI3K signalling. miRNAs have the capacity to control RNA turnover as a gene expression mechanism, and RNA stability profiling is an excellent tool for interrogating functionally relevant gene regulatory pathways and miRNA targets when combined with other high-throughput and in silico approaches.

SUBMITTER: Joshi P

PROVIDER: S-EPMC6779404 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Transcriptome stability profiling using 5'-bromouridine IP chase (BRIC-seq) identifies novel and functional microRNA targets in human melanoma cells.

Joshi Piyush P Seki Tatsuya T Kitamura Shinobu S Bergano Andrea A Lee Bongyong B Perera Ranjan J RJ

RNA biology 20190616 10

RNA half-life is closely related to its cellular physiological function, so stability determinants may have regulatory functions. Micro(mi)RNAs have primarily been studied with respect to post-transcriptional mRNA regulation and target degradation. Here we study the impact of the tumour suppressive melanoma miRNA miR-211 on transcriptome stability and phenotype in the non-pigmented melanoma cell line, A375. Using 5'-bromouridine IP chase (BRIC)-seq, transcriptome-wide RNA stability profiles reve ...[more]

PMID: 31179855

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Project description:The identification of novel tumor-specific markers may improve understanding of melanoma progression and prognostic accuracy. Whole genome expression profiling of 46 primary melanomas, 12 metastases, and 16 normal skin samples using Affymetrix U133 PLUS 2.0 array generated gene lists including both known and new melanoma genes. The molecular genetic alterations contributing to the pathogenesis of melanoma are incompletely defined and few independent prognostic features have been identified beyond the pathologic characteristics of the primary tumor. Early stage melanoma is frequently curable, in contrast to the inferior prognosis of melanomas with regional lymph nodes involvement and the incurability of distant metastatic disease. The identification of novel tumor-specific markers may improve our understanding of melanoma progression and prognostic accuracy. To find differentially expressed genes that can distinguish melanoma from normal tissue, we performed a whole genome expression profiling of 46 primary melanoma samples, 12 regional or distant metastases, and 16 normal skin samples using Affymetrix U133 2.0 Plus array. Our study generated lists of differentially expressed genes in melanoma and identified novel prognostic marker HMGA2. It is a novel, highly overexpressed melanoma gene associated with poor prognosis. The overexpression of HMGA2 is strongly associated with regional and distant metastases and serves as an independent predictor of disease-free survival and overall survival in melanoma. Melanoma samples were obtained through an IRB-approved protocol using informed consent at the University of Michigan Multidisciplinary Melanoma Clinic. A portion of pigmented lesions clinically suspicious for melanoma and known melanoma were sampled by punch biopsy at the time of excision. The punch biopsy was bisected; half was sent to for clinical diagnosis and the other half along with adjacent normal skin when available immediately snap-frozen in liquid nitrogen. Snap-frozen tissue was embedded in OCT (TissueTek) followed by frozen sectioning and H&E slide preparation. The slides were evaluated by dermatopathologist who identified areas with greater than 70% tumor cellularity, which were sampled for RNA extraction. Primary melanomas and melanoma metastases were derived from different patients. Metastatic samples included lymph nodes (n=8), subcutaneous soft tissue (n=2), spleen (n=1), and small intestine (n=1).

Project description:MicroRNAs are small RNAs that regulate gene expression through complementary base pairing with their target mRNAs. A substantial understanding of microRNA target recognition and repression mechanisms has been reached using diverse empirical and bioinformatic approaches, primarily in vitro biochemical or cell culture perturbation settings. We sought to determine if rules of microRNA target efficacy could be inferred from extensive gene expression data of human tissues. A transcriptome-wide assessment of all the microRNA-mRNA canonical interactions' efficacy was performed using a normalized Spearman correlation (Z-score) between the abundance of the transcripts in the PRAD-TCGA dataset tissues (RNA-seq mRNAs and small RNA-seq for microRNAs, 546 samples). Using the Z-score of correlation as a surrogate marker of microRNA target efficacy, we confirmed hallmarks of microRNAs, such as repression of their targets, the hierarchy of preference for gene regions (3'UTR > CDS > 5'UTR), and seed length (6 mer < 7 mer < 8 mer), as well as the contribution of the 3'-supplementary pairing at nucleotides 13-16 of the microRNA. Interactions mediated by 6 mer + supplementary showed similar inferred repression as 7 mer sites, suggesting that the 6 mer + supplementary sites may be relevant in vivo. However, aggregated 7 mer-A1 seeds appear more repressive than 7 mer-m8 seeds, while similar when pairing possibilities at the 3'-supplementary sites. We then examined the 3'-supplementary pairing using 39 microRNAs with Z-score-inferred repressive 3'-supplementary interactions. The approach was sensitive to the offset of the bridge between seed and 3'-supplementary pairing sites, and the pattern of offset-associated repression found supports previous findings. The 39 microRNAs with effective repressive 3'supplementary sites show low GC content at positions 13-16. Our study suggests that the transcriptome-wide analysis of microRNA-mRNA correlations may uncover hints of microRNA targeting determinants. Finally, we provide a bioinformatic tool to identify microRNA-mRNA candidate interactions based on the sequence complementarity of the seed and 3'-supplementary regions.

Dataset Information

Transcriptome stability profiling using 5'-bromouridine IP chase (BRIC-seq) identifies novel and functional microRNA targets in human melanoma cells.

Publications

Transcriptome stability profiling using 5'-bromouridine IP chase (BRIC-seq) identifies novel and functional microRNA targets in human melanoma cells.

Similar Datasets

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