Overexpression of miR-10a and miR-375 and downregulation of YAP1 in medullary thyroid carcinoma

https://doi.org/10.1016/j.yexmp.2013.05.001Get rights and content

Highlights

  • We examined the expression of over 700 miRNAs in medullary thyroid carcinoma (MTC).

  • miR-375 and miR-10a were overexpressed and miR-455 was underexpressed in MTC.

  • A known target of miR-375 and a growth inhibitor, YAP1, was downregulated in MTC.

  • Inhibition of YAP1 by miR-375 may be important for MTC development or progression.

Abstract

MicroRNAs are a primordial mechanism of gene expression control that appear to be crucial to cellular development and may play an important role in tumor development. Much is known about the genetics of medullary thyroid carcinomas, as approximately 25% are hereditary and harbor germ line activating mutations in the RET gene. Somatic RET mutations are also seen in roughly 50% of sporadic medullary thyroid carcinomas. Few studies, however, have evaluated the role of microRNA expression in these tumors. DNA and RNA were extracted from formalin-fixed paraffin-embedded tissue blocks of 15 medullary thyroid carcinomas [10 with RET mutations (3 hereditary) and 5 without RET mutations] and 5 non-tumor thyroid glands. miRNA expression of 754 targets was quantitated by real-time PCR using the ABI OpenArray miRNA assay. Three miRNAs showed significant differential expression and were validated in a larger cohort of 59 cases by real-time PCR. Expression of potential downstream targets and upstream regulators was also investigated by real-time PCR. miR-375 and miR-10a were significantly overexpressed, while miR-455 was underexpressed in medullary thyroid carcinomas. Expression of all 3 miRNAs was validated in the larger cohort of cases (miR-375, p = 3.3 × 10 26; miR-10a, p = 5.6 × 10 14; miR-455, p = 2.4 × 10 4). No significant differences in miRNA expression were found between RET mutation positive and negative tumors nor between sporadic and hereditary tumors. Expression of the potential downstream targets of miR-375, YAP1 (a growth inhibitor) and SLC16a2 (a transporter of thyroid hormone), was down‐regulated in the tumors suggesting that miR-375 is a negative regulator of the expression of these genes. Thus, differential expression of miR-375, miR-10a and miR-455 may be important for tumor development and/or reflect C-cell lineage of medullary thyroid carcinoma. Furthermore, the growth inhibitor YAP1 is identified as a potential important downstream target of miR-375.

Introduction

Medullary thyroid carcinoma is a neuroendocrine tumor with parafollicular (C-cell) differentiation that accounts for approximately 5% of all primary thyroid malignancies. It occurs in both hereditary (25%) and sporadic (75%) forms (Albores-Saavedra et al., 1985). Activating mutations in the RET proto-oncogene are responsible for hereditary medullary thyroid carcinoma (MEN2 syndrome and familial medullary thyroid carcinoma) (Carlson et al., 1994). Approximately 50% of sporadic cases also harbor activating RET mutations and, more recently, a subset of RET mutation negative cases have been shown to harbor RAS mutations (Boichard et al., 2012, Moura et al., 2011, Wells and Santoro, 2009). Although medullary thyroid carcinoma accounts for a small percentage of primary thyroid tumors, it is responsible for a disproportionately large number of thyroid cancer deaths due to its more aggressive behavior compared to well-differentiated papillary thyroid and follicular carcinomas. Overall, medullary thyroid carcinoma has a 10 year survival rate of 80% compared to 92% and 90% for papillary and follicular carcinomas, respectively (Gilliland et al., 1997). The prognosis of medullary thyroid carcinoma can be predicted by the particular RET mutation and also depends on stage at presentation (Gilliland et al., 1997, Modigliani et al., 1998). Locally advanced tumors and the presence of metastatic disease are associated with a 5-year survival of 40% or less (Modigliani et al., 1998).

MicroRNAs (miRNAs) are endogenous short single-stranded non-coding RNAs that are negative regulators of gene expression. They selectively bind to complementary 3′ UTR mRNAs and target them for either cleavage or translational repression (Zeng et al., 2003). To date, over 1000 human miRNAs have been identified. Although their functions have not been fully characterized, miRNAs are known to have important roles in regulating cell differentiation, proliferation and survival. It is estimated that 30% of human genes may be targeted by miRNAs (Lewis et al., 2005; Liu and Xu, 2011, Xie et al., 2005). Furthermore, many studies have supported a role for miRNAs in human cancers (Bottoni et al., 2005; Croce, 2006, Garzon et al., 2006, Kumar et al., 2008, Lu et al., 2005; Medina et al., 2010, Takamizawa et al., 2004, Zhang et al., 2006). Through negative regulation of gene expression, miRNAs can function either as ʻoncomiRsʼ to promote tumor growth and progression or as tumor suppressors (He et al., 2005). Altered miRNA expression has been detected in a variety of human cancers including esophageal, breast, gastric, colorectal, pancreatic and lung carcinomas (Lu et al., 2005, Nikitina et al., 2012).

Few studies have examined the role of miRNAs in medullary thyroid carcinoma (Abraham et al., 2011, Mian et al., 2012, Nikiforova et al., 2008). To date, only two miRNA array profiling studies of medullary thyroid carcinomas have been published (Abraham et al., 2011, Mian et al., 2012). Understanding miRNA expression patterns and their effects on gene expression may provide a better understanding of tumor development and progression and may also yield potential therapeutic targets. In this study, we evaluated the expression of over 700 miRNAs in a retrospective cohort of medullary thyroid carcinomas and found significant up-regulation of three key miRNAs compared to normal thyroid tissue. Several predicated targets of these miRNAs were evaluated and showed significant downregulation, consistent with the effects of miRNA expression. These data suggest that the expression of specific miRNAs is involved in medullary thyroid carcinoma, providing new insights to pathogenesis and possible treatment targets.

Section snippets

Case selection

The study was approved by the Human Research Protection Office of Washington University and the Institutional Review Board at the National Institute of Health. Archival, formalin-fixed, paraffin-embedded tissue blocks and slides from all available cases of medullary thyroid carcinoma from 1989 to 2009 were retrieved from the Department of Pathology and Immunology at the Washington University School of Medicine (St. Louis, MO). The hematoxylin and eosin (H&E)-stained sections were reviewed by

Results

Seventy-one human miRNAs were differently expressed in medullary thyroid carcinoma compared to non-tumor thyroid tissue on the ABI OpenArray miRNA based on a p-value cut off of 0.05. The top 10 differentially expressed genes are shown in Table 2. However, after multiple comparison correction, only three microRNAs retained statistical significance between medullary thyroid carcinoma and non-tumor thyroid tissue, including miR-375 (485 fold increase in expression, p = 0.0003), miR-10a (23 fold

Discussion

The role of miRNAs in the development and progression of medullary thyroid carcinomas has not been well examined. To our knowledge, only two other array-based studies have evaluated miRNA expression in medullary thyroid carcinoma (Lu et al., 2005, Xie et al., 2005). Nikiforova et al. (2008) were the first to investigate miRNA expression in 2 cases of medullary thyroid carcinoma as part of a larger study of all types of thyroid tumors (Nikiforova et al., 2008). Although only 2 cases were

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgments

We thank Xiaopei Zhu M.D. for technical assistance with the PCR experiments and James S Lewis Jr. for critical review of the manuscript. We also thank Chris Sawyer and the Washington University Genome Technology Access Core (GTAC) for their assistance in running the PCR arrays. The GTAC is partially supported by NCI Cancer Center support grant #P30 CA91842 to the Siteman Cancer Center and by ICTS/CTSA grant #UL1RR024992 from the National Center for Research Resources (NCRR), a component of the

References (33)

  • R. Garzon et al.

    MicroRNA expression and function in cancer

    Trends in Molecular Medicine

    (2006)
  • B.P. Lewis et al.

    Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets

    Cell

    (2005)
  • D. Abraham et al.

    MicroRNA profiling of sporadic and hereditary medullary thyroid cancer identifies predictors of nodal metastasis, prognosis, and potential therapeutic targets

    Clinical Cancer Research

    (2011)
  • J. Albores-Saavedra et al.

    Medullary carcinoma

    Seminars in Diagnostic Pathology

    (1985)
  • A. Boichard et al.

    Somatic RAS mutations occur in a large proportion of sporadic RET-negative medullary thyroid carcinomas and extend to a previously unidentified exon

    The Journal of Clinical Endocrinology and Metabolism

    (2012)
  • A. Bottoni et al.

    miR-15a and miR-16-1 down-regulation in pituitary adenomas

    Journal of Cellular Physiology

    (2005)
  • K.M. Carlson et al.

    Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B

    Proceedings of the National Academy of Sciences of the United States of America

    (1994)
  • C. Croce

    MicroRNAs in leukemia

    Clinical Advances in Hematology & Oncology

    (2006)
  • P. De Souza Rocha Simonini et al.

    Epigenetically deregulated microRNA-375 is involved in a positive feedback loop with estrogen receptor alpha in breast cancer cells

    Cancer Research

    (2010)
  • N.H. Foley et al.

    MicroRNAs 10a and 10b are potent inducers of neuroblastoma cell differentiation through targeting of nuclear receptor corepressor 2

    Cell Death and Differentiation

    (2011)
  • F.D. Gilliland et al.

    Prognostic factors for thyroid carcinoma. A population-based study of 15,698 cases from the Surveillance, Epidemiology and End Results (SEER) program 1973–1991

    Cancer

    (1997)
  • L. He et al.

    A microRNA polycistron as a potential human oncogene

    Nature

    (2005)
  • A. Kinne et al.

    Primary and secondary thyroid hormone transporters

    Thyroid Research

    (2011)
  • M.S. Kumar et al.

    Suppression of non-small cell lung tumor development by the let-7 microRNA family

    Proceedings of the National Academy of Sciences of the United States of America

    (2008)
  • N.-K. Liu et al.

    MicroRNA in central nervous system trauma and degenerative disorders

    Physiological Genomics

    (2011)
  • J. Liu et al.

    MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies

    Nature Cell Biology

    (2005)
  • Cited by (0)

    View full text