SUMMARYAs only 60% of the patients with germ cell tumour (GCT) express the classical markers, new markers as for example microRNAs (miRNAs) are required. One promising candidate is miR-371a-3p, but data are sparse to date. We measured serum levels of miR371a-3p in GCT patients, in controls, and in cases with other malignancies. We also assessed the expression in other body fluids and we looked to the decline of serum miR-371a-3p levels after treatment. miR-371a-3p levels were measured by quantitative polymerase chain reaction in serum samples of 25 GCT patients, 6 testicular intraepithelial neoplasia (TIN) patients, 20 healthy males and 24 non-testicular malignancies (NTMs). Testicular vein blood (TVB) was examined in five GCT patients and five controls. Five GCT patients had serial daily measurements after orchiectomy. Five seminal plasma samples, three urine specimens and one pleural effusion fluid were processed likewise. GCT patients had significantly higher miR-371a-3p serum levels than controls and NTMs. Serum levels of controls, TINs and NTMs were not significantly different. TVB samples of GCT patients had 65.4-fold higher serum levels than peripheral blood. Malignant pleural effusion fluid had extremely high levels of miR-371a-3p, seminal plasma had strongly elevated levels by comparison with serum levels of controls. In urine of GCT patients, no miR-371a-3p expression was detected. Daily measurements after orchiectomy in stage 1 patients revealed a decline by 95% within 24 h. Serum levels of miR-371a-3p appear to be a promising specific biomarker of GCTs as is suggested by high serum levels in GCT patients, the rapid return of elevated levels to normal range after treatment, the association of serum levels with tumour bulk, the non-expression in NTMs and the much higher levels of miR-371a-3p in TVB. This potential marker deserves further exploration in a large-scale clinical study.

HMGA2 is a major regulator of benign tumorigenesis from mesenchyme-derived tissues and stem-cell self-renewal. It has been postulated that HMGA2 mediates its critical function by decreasing p16(Ink4a)/p14(Arf) expression and cellular senescence. To repress the oncogenic activity of HMGA2, the lin-28-let-7 axis is thought to increasingly repress the expression of HMGA2 with age. To understand the HMGA2-p14(Arf) -relationship in benign tumorigenesis, we performed a series of experiments on mesenchymal stem-cells, i.e., the proposed cells of origin of lipomas and uterine leiomyomas. The expression of both genes was inversely correlated during senescence in vitro but contrary to the expectations in adipose tissue derived stem cells (ADSCs) stimulation of HMGA2 by FGF1 increased the expression of p14(Arf) . Based on the assumption that in ADSCs p14(Arf) is repressing HMGA2, siRNA silencing of p14(Arf) was performed resulting in a significant upregulation of HMGA2. To see if p14(Arf) can repress HMGA2 by a TP53-dependent mechanism, nutlin-3, a known MDM2 antagonist, was used which not only increased the activity of the senescence, associated markers p21 and beta-galactosidase, but also decreased the expression of HMGA2, suggesting that p14(Arf) indeed influences HMGA2 by a p53-dependent mechanism because nutlin-3 stabilizes p53. Accordingly, the HMGA2 response triggered by serum was reduced by treatment of ADSCs with nutlin-3. As to the interaction between HMGA2 and p14(Arf) in benign tumorigenesis, we propose a model where akin to MSC self-renewal during tissue repair the simultaneous increase of p14(Arf) with HMGA2 ensures genomic stability, whereas in turn p14(Arf) can repress HMGA2 via TP53.

Background HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure two consensus sequences for HMGA2 binding have been identified.Methodology/Principal FindingsIn this investigation chromatin immunoprecipitation (ChIP) experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well.ConclusionAfter quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp) of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.

Our data clearly show that cells of the amniotic fluid strongly overexpress HMGA2 according to their fetal origin. The fact that apparently HMGA2 remains to be attached to cell-free DNA suggests interesting new approaches in noninvasive prenatal diagnosis.