The Two Stem Cell MicroRNA Gene Clusters C19MC and miR-371-3 Are Activated by Specific Chromosomal Rearrangements in a Subgroup of Thyroid Adenomas

Thyroid adenomas are common benign human tumors with a high prevalence of about 5% of the adult population even in iodine sufficient areas. Rearrangements of chromosomal band 19q13.4 represent a frequent clonal cytogenetic deviation in these tumors making them the most frequent non-random chromosomal translocations in human epithelial tumors at all. Two microRNA (miRNA) gene clusters i.e. C19MC and miR-371-3 are located in close proximity to the breakpoint region of these chromosomal rearrangements and have been checked for a possible up-regulation due to the genomic alteration. In 4/5 cell lines established from thyroid adenomas with 19q13.4 rearrangements and 5/5 primary adenomas with that type of rearrangement both the C19MC and miR-371-3 cluster were found to be significantly overexpressed compared to controls lacking that particular chromosome abnormality. In the remaining cell line qRT-PCR revealed overexpression of members of the miR-371-3 cluster only which might be due to a deletion accompanying the chromosomal rearrangement in that case. In depth molecular characterization of the breakpoint in a cell line from one adenoma of this type reveals the existence of large Pol-II mRNA fragments as the most likely source of up-regulation of the C19MC cluster. The up-regulation of the clusters is likely to be causally associated with the pathogenesis of the corresponding tumors. Of note, the expression of miRNAs miR-520c and miR-373 is known to characterize stem cells and in terms of molecular oncology has been implicated in invasive growth of epithelial cells in vitro and in vivo thus allowing to delineate a distinct molecular subtype of thyroid adenomas. Besides thyroid adenomas rearrangements of 19q13.4 are frequently found in other human neoplasias as well, suggesting that activation of both clusters might be a more general phenomenon in human neoplasias

Delineation of <i>PUM1</i> breakpoint by metaphase FISH.

<p>Part of metaphase of cell line S40.2 after FISH with two overlapping BAC clones RP11-201O14 (green) and RP11-1136E4 (red) both spanning the whole genomic sequence of <i>PUM1</i> in 1p35.2. The breakpoint in 1p35.2 is located within <i>PUM1</i> indicated by a separation of RP11-201O14 and RP11-1136E4. Because of weak signals of RP11-1136E4 remaining on the der(1) the breakpoint is located within RP11-1136E4 distal to RP11-201O14.</p

Fluorescence in situ hybridization (FISH) with dual-color, break-apart rearrangement probe (tbpc19).

<p>(<b>A</b>)<b>–</b>(<b>E</b>) I-FISH showing 19q13 rearrangements detected using touch-preparations of five thyroid adenomas (<b>A:</b> S801, <b>B:</b> S814, <b>C:</b> S842, <b>D:</b> S846, <b>E:</b> S849) indicated by separated green (3′-tbpc19) and red signals (5′-tbpc19); (<b>F</b>) Metaphase of case S842 with a t(1;19)(q32;q13) after FISH with tbpc19. The 19q13 rearrangement is indicated also by separated signals on der(1) and der(19).</p

Scheme of the chromosomal region 19q13.4 with the two miRNA clusters C19MC and miR-371-3.

<p>Protein coding genes are represented by gray bars whereas genes of miRNA clusters are given as blue (C19MC cluster) and green (miR-371-3 cluster) lines, respectively. The common breakpoint cluster (BPC) of benign thyroid tumors of about 150 kb is indicated by a vertical arrow. miR-512-1 (pre-miR) is coding for mature-miR-512-5p, miR-371 (pre-miR) is coding for mature-miR371-3p. Gene symbols refer to the following protein coding genes: <i>ZNF331</i>  =  <i>zinc finger protein 331</i>, <i>DPRX</i>  =  <i>divergent-paired related homeobox</i>, <i>NLRP12</i>  =  <i>NLR family</i>, <i>pyrin domain containing 12.</i></p

Used Primers.

<p>Primer sequences for PCR and cDNA synthesis.</p

Used tissue samples and cell lines.

<p>Cytogenetic details of the analyzed samples from follicular thyroid tumors and the cell lines used with their genetic subgroups determined by conventional cytogenetics and/or by interphase fluorescence in situ hybridization (I-FISH) with break-apart, dual-color rearrangement probe (tbpc-19). In case of the cell lines only the clonal aberrations found in the original tumors the cell lines have been established from are given.</p><p>Ref.: (_*) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009485#pone.0009485-Belge4" target="_blank">[30]</a>; (#) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009485#pone.0009485-Belge1" target="_blank">[2]</a>; (π) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009485#pone.0009485-Bol1" target="_blank">[31]</a>.</p

Partial karyotype of cell line S40.2.

<p>Partial G-banded karyotype showing chromosome 1 and 19 as well as their derivatives resulting from t(1;19)(p35.2;q13.4).</p

Statistical analysis of the qRT-PCR data.

<p>Statistical analysis (t-test, two-tailed) of the expression of miRNAs from the cluster C19MC and miR-371-3 in tissues or cell lines containing 19q13 rearrangements compared to normal thyroid tissue and/or adenomas without 19q13 rearrangements. The data were obtained using statistical software R (<a href="http://www.r-project.org" target="_blank">www.r-project.org</a>). (d.f. =  degrees of freedom, t =  Student's t-value)</p

Genomic organization of the fusion gene on the derivative chromosome 1 resulting from a translocation t(1;19)(p35.2;13.4) in cell line S40.2.

<p>Detailed schematic overview illustrating the origin of the fusion transcripts <i>PUM1-FUS-19q-I</i> (Genbank Accession number GQ334687) and PUM1-FUS-19q-II (Genbank Accession number GQ334688) identified in cell line S40.2. The genomic region of PUM1 in 1p35.2 (horizontal gray bar) fuses after exon 10 of <i>PUM1</i> (exons: vertical light gray bars) to the genomic region of <i>C19MC</i> in 19q13.4 (horizontal red bar). The two vertical yellow bars indicate 3′-sequences located after exon 1–10 of <i>PUM1</i> in <i>PUM1-FUS-19q-I</i> and <i>PUM1-FUS-19q-II</i>, respectively, both originating from alternative splicing. The fusion transcripts were detected either by 3′-RACE-PCR (<i>PUM1-FUS-19q-I</i>) or RT-PCR (<i>PUM1-FUS-19q-II</i>) experiments. The quantified miRNAs have been highlighted by their names.</p