Integrative testis transcriptome analysis reveals differentially expressed miRNAs and their mRNA targets during early puberty in Atlantic salmon.

BACKGROUND: Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. RESULTS: Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. CONCLUSIONS: This study has - for the first time - linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis.

Highly infiltrative brain tumours show reduced chemosensitivity associated with a stem cell-like phenotype.

AIMS: Cancer stem-like cells might have important functions in chemoresistance. We have developed a model where highly infiltrative brain tumours with a stem-like phenotype were established by orthotopic transplantation of human glioblastomas to immunodeficient rats. Serial passaging gradually transformed the tumours into a less invasive and more angiogenic phenotype (high-generation tumours). The invasive phenotype (low-generation tumours) was characterized by an increase in stem cell markers and increased phosphorylation of kinases in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. These markers were reduced in the serially passaged vascular tumours. The present study was aimed at investigating how the two phenotypes responded in vitro to doxorubicin, a clinically potent cytotoxic drug for solid tumours. METHODS: Biopsy spheroids were implanted and passaged intracranially in nude rats. Gene expression and protein analyses were performed, and drug sensitivity was assessed. RESULTS: Microarray analysis revealed gene ontology categories connected to developmental aspects and negative regulators of differentiation, especially in the infiltrative stem cell-like tumours. The highly invasive stem-like phenotype was chemoresistant compared with the angiogenic phenotype. By interfering with the PI3K it was possible to sensitize tumour spheroids to chemotherapy. Real-time quantitative polymerase chain reaction showed downregulation of the stem cell markers Nestin and Musashi-1 in low-generation biopsy spheroids following PI3K inhibition. CONCLUSIONS: Highly invasive tumours with a stem-like phenotype are more chemoresistant than angiogenic tumours derived from the same patients. We suggest that treatment resistance in glioblastomas can be related to PI3K/AKT activity in stem-like tumour cells, and that targeted interference with the PI3K/AKT pathway might differentiate and sensitize this subpopulation to chemotherapy.

Primary glioma spheroids maintain tumourogenicity and essential phenotypic traits after cryopreservation.

Tumour spheroids initiated from glioma biopsy specimens provide a valuable three-dimensional cell culture system that share several biological features of malignant brain tumours in situ. Upon xenotransplantation in immunodeficient rats, tumours derived from such spheroids exhibit a highly infiltrative growth. Successful cryopreservation of spheroid specimens therefore represents an excellent tool for future comparative studies of tumour growth and progression. Thus, if frozen stocks of human glioma spheroids can be established, similar to those obtained from cancer cell lines, it would ease the planning of biopsy-based experiments. In this context, it is crucial that cryopreservation does not alter the biological behaviour of the tumour spheroids. The biopsy spheroids were frozen to -40 degrees Celsius, stored for 1 week at -196 degrees Celsius, thawed rapidly and cultured for 1 week. The viability of the spheroids was compared against controls using a two-colour fluorescence assay, which demonstrated that cryopreservation was well tolerated. Using an in vitro invasion assay, it is shown that the freezing procedures did not affect the spheroids ability to invade a collagen gel. Cryopreserved and control tumour spheroids were equally tumourogenic, and produced overlapping survival curves when transplanted into the brains of immunocompromised rats. Immunohistochemical analyses showed no significant changes regarding microvessel density or proliferation index. Furthermore, gene expression profiling using a macroarray system detected no significant changes following cryopreservation. The present data show that cryopreservation is well tolerated, and represent a methodologically reliable storage method for biopsy spheroids that can be used in experimental studies at later time points.

Molecular approaches to brain tumour invasion.

In glioma cells, the stimulatory input of extracellular matrix components and an increased sensitivity to growth factors result in a high proliferative and migratory behaviour. Cell surface receptor interactions play pivotal roles in converging information about conditions in the environment immediately outside the cell. The transduced signal, in turn induces a response within the cell that provokes a specific behaviour. Cellular migration and cell proliferation are interwoven processes that share several common intracellular pathways. The major cross-links are the phosphoinositol phosphate regulating enzymes, PI-3 kinase and PTEN, the focal adhesion kinase (FAK) and the tumour suppressor p53. An understanding of the interaction between the molecular participants involved in migration and proliferation will promote the design of new treatments. A full understanding of the basis of the invasiveness of tumour cells remains elusive. Gene and protein expression are being studied, using modern techniques such as microarray analysis, SAGE and 2-D protein gels. Transient and permanent protein-protein interactions and recruitment of proteins to specialised cellular domains are equally important in regulating cellular invasion and presumably will attract more attention in future.