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Regulation of nucleolin expression by miR-194, miR-206, and HuR

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Abstract

Nucleolin is a proliferation-associated protein that is overexpressed in multiple types of cancer. The mechanisms leading to overexpression of nucleolin in specific cancers are not fully understood. This study found that nucleolin is notably elevated in breast cancer cell lines MCF-7 and MDA-231 compared to nonmalignant breast epithelial MCF-10A cells. In silico analyses revealed the presence of putative binding sites for microRNAs miR-194 and miR-206 in the 3′-untranslated region (3′-UTR) of Ncl mRNA. Transfection of the three cell lines with pre-miR-194 or pre-miR-206 specifically decreased the Ncl mRNA and protein expression. Treatments of the cells with antagomiR-194 or antagomiR-206 upregulated nucleolin expression ~2- to 3-fold. Co-transfection of cells with a reporter vector containing the Ncl 3′-UTR downstream from the Renilla luciferase gene and pre-miR-194 or pre-miR-206 led to a ~3-fold decrease in Renilla/firefly luciferase activity. Cytoplasmic levels of the RNA-binding protein HuR were higher in MCF-7 and MDA-231 cells than those in MCF-10A cells. RNA immunoprecipitation assays demonstrated that HuR binds to Ncl mRNA in all the three cell types. ShRNA-mediated knock-down of HuR induced a decrease in nucleolin expression, while exogenous expression of HuR led to upregulation of nucleolin expression. Analysis of the polysome–monosome distribution of Ncl mRNA in HuR knock-down cells demonstrated that HuR enhances the translation efficiency of Ncl mRNA. These findings demonstrate that nucleolin expression is down-regulated by miR-194 and miR-206 and upregulated by HuR.

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Abbreviations

miRNA:

MicroRNA

HuR:

Human antigen R

shRNA:

Short hairpin interfering RNA

Ncl:

Nucleolin

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Acknowledgments

The authors thank Dr. Daniella Ishimaru for analysis of miR binding sites in Ncl mRNA.

Funding

This work was supported by the National Cancer Institute Grant CA 87553 (to E.K.S.), MUSC Health Science Foundation Liver Research Funds (A.R.), and American Heart Association Grant 05554700 (B.G.T.). T.E.T. was supported by a fellowship from the MUSC Summer Health Research Program. Imaging facilities were supported, in part, by Cancer Center Support Grant P30 CA138313 to the Hollings Cancer Center (MUSC).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, P. O. Box 250509, Charleston, SC, 29425, USA

    Sudeep Bose, Baby G. Tholanikunnel & Eleanor K. Spicer

  2. College of Medicine, Medical University of South Carolina, Charleston, SC, 29425, USA

    Tracy E. Tholanikunnel

  3. Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, Charleston, SC, 29425, USA

    Adrian Reuben

  4. Amity Centre for Medical Biotechnology, Amity Institute of Biotechnology, Amity University, Gautam Buddha Nagar Sec-125, Noida, 201301, India

    Sudeep Bose

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  1. Sudeep Bose
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Correspondence to Baby G. Tholanikunnel.

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Bose, S., Tholanikunnel, T.E., Reuben, A. et al. Regulation of nucleolin expression by miR-194, miR-206, and HuR. Mol Cell Biochem 417, 141–153 (2016). https://doi.org/10.1007/s11010-016-2721-2

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  • DOI: https://doi.org/10.1007/s11010-016-2721-2

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