Testes and brain gene expression in precocious male and adult maturing Atlantic salmon (Salmo salar).

BACKGROUND: The male Atlantic salmon generally matures in fresh water upon returning after one or several years at sea. Some fast-growing male parr develop an alternative life strategy where they sexually mature before migrating to the oceans. These so called 'precocious' parr or 'sneakers' can successfully fertilise adult female eggs and so perpetuate their line. We have used a custom-built cDNA microarray to investigate gene expression changes occurring in the salmon gonad and brain associated with precocious maturation. The microarray has been populated with genes selected specifically for involvement in sexual maturation (precocious and adult) and in the parr-smolt transformation. RESULTS: Immature and mature parr collected from a hatchery-reared stock in January were significantly different in weight, length and condition factor. Changes in brain expression were small - never more than 2-fold on the microarray, and down-regulation of genes was much more pronounced than up-regulation. Significantly changing genes included isotocin, vasotocin, cathepsin D, anamorsin and apolipoprotein E. Much greater changes in expression were seen in the testes. Among those genes in the testis with the most significant changes in expression were anti-Mullerian hormone, collagen 1A, and zinc finger protein (Zic1), which were down-regulated in precocity and apolipoproteins E and C-1, lipoprotein lipase and anti-leukoproteinase precursor which were up-regulated in precocity. Expression changes of several genes were confirmed in individual fish by quantitative PCR and several genes (anti-Mullerian hormone, collagen 1A, beta-globin and guanine nucleotide binding protein (G protein) beta polypeptide 2-like 1 (GNB2L1) were also examined in adult maturing testes. Down-regulation of anti-Mullerian hormone was judged to be greater than 160-fold for precocious males and greater than 230-fold for November adult testes in comparison to July testes by this method. For anti-Mullerian hormone and guanine nucleotide binding protein beta polypeptide 2-like 1 expression changes in precocious males mirrored mature adults (November) but for collagen 1A and beta-globin the pattern was more complex. CONCLUSIONS: Expression changes in the fish brain during the process of precocious sexual maturation were small compared to those in the testes. Microarray analysis suggested down-regulation of housekeeping functions and up-regulation of a small number of specific processes. Transcriptional changes in the testes were much more pronounced with anti-Mullerian hormone playing a major role. Expression profiles for mature parr and maturing adult testes indicate subtle differences in gene expression between these two related groups.

MicroRNA signatures predict oestrogen receptor, progesterone receptor and HER2/neu receptor status in breast cancer.

INTRODUCTION: Breast cancer is a heterogeneous disease encompassing a number of phenotypically diverse tumours. Expression levels of the oestrogen, progesterone and HER2/neu receptors which characterize clinically distinct breast tumours have been shown to change during disease progression and in response to systemic therapies. Mi(cro)RNAs play critical roles in diverse biological processes and are aberrantly expressed in several human neoplasms including breast cancer, where they function as regulators of tumour behaviour and progression. The aims of this study were to identify miRNA signatures that accurately predict the oestrogen receptor (ER), progesterone receptor (PR) and HER2/neu receptor status of breast cancer patients to provide insight into the regulation of breast cancer phenotypes and progression. METHODS: Expression profiling of 453 miRNAs was performed in 29 early-stage breast cancer specimens. miRNA signatures associated with ER, PR and HER2/neu status were generated using artificial neural networks (ANN), and expression of specific miRNAs was validated using RQ-PCR. RESULTS: Stepwise ANN analysis identified predictive miRNA signatures corresponding with oestrogen (miR-342, miR-299, miR-217, miR-190, miR-135b, miR-218), progesterone (miR-520g, miR-377, miR-527-518a, miR-520f-520c) and HER2/neu (miR-520d, miR-181c, miR-302c, miR-376b, miR-30e) receptor status. MiR-342 and miR-520g expression was further analysed in 95 breast tumours. MiR-342 expression was highest in ER and HER2/neu-positive luminal B tumours and lowest in triple-negative tumours. MiR-520g expression was elevated in ER and PR-negative tumours. CONCLUSIONS: This study demonstrates that ANN analysis reliably identifies biologically relevant miRNAs associated with specific breast cancer phenotypes. The association of specific miRNAs with ER, PR and HER2/neu status indicates a role for these miRNAs in disease classification of breast cancer. Decreased expression of miR-342 in the therapeutically challenging triple-negative breast tumours, increased miR-342 expression in the luminal B tumours, and downregulated miR-520g in ER and PR-positive tumours indicates that not only is dysregulated miRNA expression a marker for poorer prognosis breast cancer, but that it could also present an attractive target for therapeutic intervention.

Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer.

The discovery of microRNAs (miRNAs) added an extra level of intricacy to the already complex system regulating gene expression. These single-stranded RNA molecules, 18-25 nucleotides in length, negatively regulate gene expression through translational inhibition or mRNA cleavage. The discovery that aberrant expression of specific miRNAs contributes to human disease has fueled much interest in profiling the expression of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalised to an endogenous control (EC) gene, which ideally, is stably-expressed across the test sample set. A universal endogenous control suitable for every tissue type, treatment and disease stage has not been identified and is unlikely to exist, so, to avoid introducing further error in the quantification of expression data it is necessary that candidate ECs be validated in the samples of interest. While ECs have been validated for quantification of mRNA expression in various experimental settings, to date there is no report of the validation of miRNA ECs for expression profiling in breast tissue. In this study, the expression of five miRNA genes (let-7a, miR-10b, miR-16, miR-21 and miR-26b) and three small nucleolar RNA genes (RNU19, RNU48 and Z30) was examined across malignant, benign and normal breast tissues to determine the most appropriate normalisation strategy. This is the first study to identify reliable ECs for analysis of miRNA by RQ-PCR in human breast tissue.