Elsevier

Cancer Genetics

Volume 208, Issue 5, May 2015, Pages 279-287
Cancer Genetics

Contributions to Progress and Promise of Epigenetics for Diagnosis and Therapy in Cancer
WEE1 is a validated target of the microRNA miR-17-92 cluster in leukemia

https://doi.org/10.1016/j.cancergen.2015.01.001Get rights and content

MicroRNAs are short single-stranded RNAs that regulate target gene expression by binding to complementary sites in the 3′ untranslated region (UTR) of their mRNA targets. The polycistronic miR-17-92 cluster, which encodes miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a, was previously shown to be overexpressed in multiple types of cancer. In this study, target gene prediction algorithms were used to predict potential targets of the miR-17-92 cluster. WEE1, a kinase that inhibits cell cycle progression, was identified as a possible target of five of the six miRNAs in the cluster. Luciferase reporter assays were used to determine that miR-17, miR-20a, and miR-18a specifically target nucleotides 465–487 of the 3′ UTR of WEE1, whereas miR-19a and miR-19b exert control on WEE1 by targeting nucleotides 1069–1091. A negative correlation was determined between endogenous miR-17 or miR-19a expression and endogenous WEE1 protein expression in the same panel of cell lines. We conclude that WEE1 is a valid target of the miR-17-92 cluster in leukemia.

Section snippets

Prediction of miRNA target genes

Prediction algorithms including TargetScan (www.targetscan.org), MicroCosm Targets (www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5), PicTar (pictar.mdc-berlin.de), and miBridge (sitemaker.umich.edu/mibridge/target_predictions) were used to predict potential biological targets of the miR-17-92 cluster.

Cloning of luciferase reporter constructs

The 3′ untranslated region (UTR) of WEE1 (nucleotides 2418–3356 of NM_003390.3) was amplified from human genomic DNA using the forward primer 5′ATGTTACACCAGCCTTTCCAGGGT3′ and the reverse

WEE1 is a predicted target of multiple miRNAs within the miR-17-92 cluster

Bioinformatic algorithms for miRNA target gene prediction were used to predict potential targets of miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. For each miRNA of the cluster, a list was compiled of the 30 target genes with the highest total context scores from TargetScan, the 20 target genes with the lowest P values from MicroCosm Targets, the 20 target genes with the highest PicTar scores from PicTar, and all predicted target genes from miBridge. Both MicroCosm Targets and

Discussion

Identifying targets of the miR-17-92 cluster is important for increased understanding of the complex gene expression pathways that are dysregulated in leukemia. Previous studies have experimentally validated a range of gene targets of the miR-17-92 cluster in various systems. The individual miRNAs of this cluster have been shown to promote cell proliferation by downregulating p21 (17) and Pten (18), suppress apoptosis by downregulating E2Fs (19) and Bim (20), and induce angiogenesis in solid

Acknowledgments

The MSCV-PIG, miR-17 MSCV-PIG, miR-17-19b MSCV-PIG, and miR-17-92 MSCV-PIG plasmids were a kind gift from Dr. Jianjun Chen at the University of Chicago. This work was supported by the National Institutes of Health grant HL087188 (NJZ-L).

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    Present address: Division of Hematology/Oncology, Department of Medicine, Northwestern University, 303 East Superior Street, Chicago, IL, USA.

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