Additional file 1: of New global analysis of the microRNA transcriptome of primary tumors and lymph node metastases of papillary thyroid cancer

Figure S1 to S9 and Table S1 to S4. (PDF 1467 kb

Additional file 2: of New global analysis of the microRNA transcriptome of primary tumors and lymph node metastases of papillary thyroid cancer

Supplemental materials and methods. (PDF 652 kb

Normalized luciferase activity in HEK293 cells cotransfected during 24 h or 48 h with the 3′UTR-LOX construction or with the mutated construction (3′UTR-LOX mutated: partial deletion of the seed sequence) and with miR-29a or with the scramble.

<p>The seed sequence is represented in blue and the * indicates a p-value<5%.</p

Proposed scheme synthetizing the miRNA and mRNA expression analyses of the same 11 ATC and highlighting the EMT feature of this tumor.

<p>Only direct binding of miRNA on mRNA are represented (green: downregulated m(i)RNA; red: upregulated m(i)RNA.</p

Heatmap based on the results of SAM one class analysis of the microarray data from 6 thyroid cancer cell lines and compared to tumor tissues.

<p>18 of the 19 modulated miRNA in cell lines are modulated in the same way in ATC, but are not modulated or in an opposite way in differentiated tumors (PTC and FTC). The heatmap was generated using R 2.14.1. with the library “gplots”. ATC.M (n = 11), PTC.M (n = 8) and FTC.M (n = 8) refer to the mean of the miRNA data from the different tumors analyzed. * miRNA described as regulated in ATC by Braun et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111581#pone.0111581-Braun1" target="_blank">[23]</a>. # miRNA described as regulated in ATC by Visone et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111581#pone.0111581-Visone1" target="_blank">[24]</a>.</p

Immunohistochemistry with the CD163 antibody on 3 different ATC and on normal thyroid tissue.

<p>a, b, c: 20× magnification showing that about 50% of the nucleated cells are TAM, d: 40× magnification showing elongated cytoplasmic extensions in TAM as illustrated by the red arrows; e: 20× magnification illustrating the almost absence of TAM in normal thyroid tissue.</p

<i>In situ</i> hybridization of Let-7g, miR-29a and miR-30e in normal and ATC tissues.

<p>Tissue sections from normal and tumor thyroids were incubated with a full length DIG-labeled LNA probe to detect Let-7g, miR-29a and miR-30e: 20× magnification showing a strong miRNA signal observed (in blue) in the normal thyrocytes and a weak signal observed in the tumor cells derived from thyrocytes. No signal was detected in the TAM. 4 ATC were investigated. Representative pictures of 3 of them are shown.</p

A: Principal Component Analysis of the miRNA expression data from primary cultures stimulated for 1,5, 3, 16, 24 and 48 hours with TSH or with EGF/serum and from tumor tissues (autonomous adenomas (AA) and papillary thyroid carcinomas (PTC)).

<p>The two main components are shown. All the treated primary cultured samples are localized close to each other near the origins of the axis, illustrating the low change on the miRNA expression resulting from the treatment. The proportion of variance explained by the Principal Component 1 and 2 are 0.353 and 0.270, respectively. The PCA was generated with R 2.8.1 with the function Prcomp. <b>B: Multi-dimensional scaling analysis (MDS) based on four independent human primary thyroid cell cultures stimulated with 25 ng/ml EGF and 10% serum (E) for 1.5, 3, 16, 24 and 48 hours and on five independent human primary thyroid cultures stimulated with 0.3 mU/ml TSH (T) for similar times <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111581#pone.0111581-vanStaveren4" target="_blank">[32]</a></b><b>.</b> Expression profiles from a pool of autonomous adenomas (AA) and of a group of papillary thyroid carcinomas (PTC) were included. The MDS is based on all the spots present on the array. The distortion of distances (stress) between the MDS 2D space and the actual gene space is 19.6%.</p

Hierarchical clustering on the miRNA expression data from primary cultures treated with TSH or with EGF/serum and from tumor tissues.

<p>Hierarchical clusterings were generated using R 2.8.1. The method used to compute the distance was “Ward”. A. Hierarchical clustering on the data from the primary cultured thyrocytes stimulated with 0.3 mU/ml of TSH for 1.5, 3, 16, 24, 48 and 72 hours and from 7 autonomous hyperfunctioning adenomas (AA). A time evolution in miRNA expression according to TSH treatment of primary cultured thyrocytes is observed suggesting fine changes in miRNA level. The AA cluster apart from the TSH treated primary cultures indicating a clear separation between those two groups of samples. Each time point is the mean of triplicates. B. Hierarchical clustering on the data from primary cultured thyrocytes stimulated with 25 ng/ml EGF and 10% serum for 1.5, 3, 16, 24, 48 and 72 hours and from 8 papillary thyroid carcinomas. In this case, no temporal evolution could be observed by hierarchical clustering. Each time point is the mean of triplicates. Tumors and treated primary cultures clustered apart, suggesting that primary cultures evolved differently according to the treatment but that there was no convergence to AA or PTC for the long-term treatment.</p

miRNA regulated in primary culture (qRT-PCR).

<p>miRNA regulated in primary culture (qRT-PCR).</p