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Review
. 2016 Mar 15;7(11):12731-47.
doi: 10.18632/oncotarget.7262.

miR-451a is underexpressed and targets AKT/mTOR pathway in papillary thyroid carcinoma

Emanuela Minna  1 , Paola Romeo  1 , Matteo Dugo  2 , Loris De Cecco  2 , Katia Todoerti  3 , Silvana Pilotti  4 , Federica Perrone  4 , Ettore Seregni  5 , Luca Agnelli  6 , Antonino Neri  6   7 , Angela Greco  1 , Maria Grazia Borrello  1
Affiliations
Review

miR-451a is underexpressed and targets AKT/mTOR pathway in papillary thyroid carcinoma

Emanuela Minna et al. Oncotarget. .

Erratum in

Abstract

Papillary Thyroid Carcinoma (PTC) is the most frequent thyroid cancer. Although several PTC-specific miRNA profiles have been reported, only few upregulated miRNAs are broadly recognized, while less consistent data are available about downregulated miRNAs. In this study we investigated miRNA deregulation in PTC by miRNA microarray, analysis of a public dataset from The Cancer Genome Atlas (TCGA), literature review and meta-analysis based on a univocal miRNA identifier derived from miRBase v21. A list of 18 miRNAs differentially expressed between PTC and normal thyroid was identified and validated in the TCGA dataset. Furthermore, we compared our signature with miRNA profiles derived from 15 studies selected from literature. Then, to select possibly functionally relevant miRNA, we integrated our miRNA signature with those from two in vitro cell models based on the PTC-driving oncogene RET/PTC1. Through this strategy, we identified commonly deregulated miRNAs, including miR-451a, which emerged also by our meta-analysis as the most frequently reported downregulated miRNA. We showed that lower expression of miR-451a correlates with aggressive clinical-pathological features of PTC as tall cell variant, advanced stage and extrathyroid extension. In addition, we demonstrated that ectopic expression of miR-451a impairs proliferation and migration of two PTC-derived cell lines, reduces the protein levels of its recognized targets MIF, c-MYC and AKT1 and attenuates AKT/mTOR pathway activation.Overall, our study provide both an updated overview of miRNA deregulation in PTC and the first functional evidence that miR-451a exerts tumor suppressor functions in this neoplasia.

Keywords: AKT pathway; RET/PTC; miR-451a; miRNA; papillary thyroid carcinoma.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest

Figures

Figure 1
Figure 1. miRNA expression profiles in PTC clinical samples
A. Heat map showing miRNAs differentially expressed between PTC and normal thyroid tissues (absolute FC≥1.5; FDR<0.05). B-C. miRNA expression in the validation set of 499 PTC and 59 normal thyroid tissues derived from TCGA. miR abundance is reported as log2 normalized (reads-per-million, RPM) median-centered; q-value, statistical significance calculated by Wilcoxon test adjusted by Benjamini-Hochberg correction.
Figure 2
Figure 2. Meta-analysis of miRNA expression profiles in PTC versus normal thyroid
Study-miRNA matrix showing miRNA expression across the 15 studies selected from literature and our study. Color code: red, upregulated; green, downregulated. Score is calculated based on the reporting frequency of each miRNA. Matrix selectively represents only miRNAs reported in at least two independent studies (complete data available in Supplemental Table S3); the three and two datasets derived from study 13 (Swierniak 2013) and from study 14 (TCGA 2014), respectively, were considered as single studies.
Figure 3
Figure 3. Combined analysis of miRNAs in PTC clinical samples and in vitro cell models
A-B. Schematic representation of the two complementary in vitro cell models based on the PTC-driving oncogene RET/PTC1. Model 1: human primary thyrocytes exogenously expressing RET/PTC1; Model 2: PTC-derived cell line TPC1 expressing endogenous RET/PTC1 treated with the RET inhibitor RPI-1. C. Venn diagram of miRNAs differentially expressed in clinical specimens (PTC vs. normal thyroid, absolute FC ≥1.5; FDR<0.05; see Supplemental Table S2) and in cell models (Model 1: RET/PTC1-expressing thyrocytes vs. parental thyrocytes, absolute FC ≥2.5; Model 2: TPC1 treated with solvent DMSO vs. TPC1 treated with RPI-1, absolute FC ≥2.5 and FDR <0.1). Common miRNAs concordantly expressed in the 3 datasets are indicated in the right boxes.
Figure 4
Figure 4. miR-451a expression relative to clinical pathological features and genetic lesions
miR-451a expression in the set of 59 normal thyroid (NT) and 499 PTC samples derived from TCGA and stratified according to A. histological type (Fv, Follicular variant; Cv, Classic variant; TCv, Tall Cell variant), B. pathological TNM stage, C. extrathyroid extension, ETE (None, absence of ETE; Min, Minimal; Mod/Adv, Moderate/Advanced), D. tumor size, E. T stage, F. N stage (N0, absence of lymph node metastases; N1 presence of lymph node metastases) and G. genetic lesion (mut, mutation; fus, fusion; Rest, samples harboring other genetic lesions). miR-451a abundance is reported as log2 normalized (reads-per-million, RPM) and represented by box plots. In parentheses the number of samples included in each group. P, statistical significance by Kruskal-Wallis test. * p-value<0.05, ** p-value<0.01 by Wilcoxon test.
Figure 5
Figure 5. miR-451a functional studies
A. miRNA-451a expression by qRT-PCR in the PTC-derived cell lines TPC1, NIM1, K1 and BCPAP, and in the normal thyroid control cells T686. Data are presented relative to the value of T686 cells. B. MIF protein expression by western blot analysis (WB) in the same panel of cell lines; Actin is shown as loading control. Below the corresponding densitometric quantification; MIF expression was normalized to Actin and presented relative to the level of T686 cells. C. Relative expression of the pair miR-451a and MIF; data are derived from the analyses reported in (A) and (B). D-I. Functional studies in NIM1 and TPC1 cells either transfected with miR-451a synthetic mimic (miR-451a) or Negative-Control (NC). D, G. Representative images of transfected cells (LEICA inverted microscope, scale bar 100μm). Cell number was determined by nuclei staining; data are presented relative to NC-transfected cells. E, H. Cell proliferation following transfection. F, I. WB analysis (day 3 after transfection). Protein expression was quantified and normalized to the loading control GAPDH. Data are presented relative to NC-transfected cells. Graphs report mean ± s.e.m. of at least two independent experiments. * p-value<0.05, ** p-value<0.005, *** p-value<0.0001 by Student's t-test.
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