Functional effect of mir-27b on myostatin expression: a relationship in Piedmontese cattle with double-muscled phenotype

BACKGROUND: In Piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point mutation in the myostatin (MSTN) gene. The Piedmontese MSTN missense mutation G938A is translated to C313Y myostatin protein. This mutation alters MSTN function as a negative regulator of muscle growth, thereby inducing muscle hypertrophy. MiRNAs could play a role in skeletal muscle hypertrophy modulation by down-regulating gene expression. RESULTS: After identifying a 3(′)-UTR consensus sequence of several negative and positive modulator genes involved in the skeletal muscle hypertrophy pathway, such as IGF1, IGF1R, PPP3CA, NFATc1, MEF2C, GSK3b, TEAD1 and MSTN, we screened miRNAs matching to it. This analysis led to the identification of miR-27b, miR-132, miR-186 and miR-199b-5p as possible candidates. We collected samples of longissimus thoracis from twenty Piedmontese and twenty Friesian male bovines. In Piedmontese group miR-27b was up-regulated 7.4-fold (p < 0.05). Further, we report that the level of MSTN mRNA was about 5-fold lower in Piedmontese cattle vs Friesian cattle (p < 0.0001) and that less mature MSTN protein was detected in the Piedmontese one (p < 0.0001). Cotransfection of miR-27b and psi-check2 vector with the luciferase reporter gene linked to the bovine wild-type 3(′)-UTR of MSTN strongly inhibited the luciferase activity (79%, p < 0.0001). CONCLUSIONS: These data demonstrate that bovine MSTN is a specific target of miR-27b and that miRNAs contribute to explain additive phenotypic hypertrophy in Piedmontese cattle selected for the MSTN gene mutation, possibly outlining a more precise genetic signature able to elucidate differences in muscle conformation

Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells

The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow

External and internal EGFR-activating signals drive mammary epithelial cells proliferation and viability

During puberty, the mammary gland undergoes an intense growth, dependent on the interplay between the Epidermal Growth Factor Receptor (EGFR) in the stroma and different mammary epithelial receptors. We hypothesize that EGFR expressed in the mammary epithelium also has a role in puberty and the epithelial cells can self-sustain by EGFR-mediated autocrine signaling. We adopted mammary cell lines from different species, as in vitro model for the epithelium, and we observed that EGFR-signaling positively affects their survival and proliferation. Once deprived of external growth factors, mammary cells still showed strong Erk 1/2 phosphorylation, abolished upon EGFR inhibition, coupled with a further reduction in survival and proliferation. Based on gene expression analysis, three EGFR-ligands (AREG, EREG and HBEGF) are likely to mediate this autocrine signaling. In conclusion, internal EGFR-activating signals sustain mammary epithelial cell proliferation and survival in vitro

MicroARNs como biomarcadores de salud y bienestar animal en cerdos

Los microARNs (miARNs) son pequeñas moléculas de ARN no codificante muy conservadas que intervienen en una amplia gama de procesos biológicos mediante la regulación postranscripcional de la expresión génica. Un aspecto intrigante en la identificación de estas moléculas como biomarcadores se deriva de su papel en la comunicación entre células, su secreción activa desde las células al medio extracelular, su alta estabilidad en los fluidos corporales y su facilidad de obtención.Todas estas características confieren a los miARNs el potencial para convertirse en una herramienta no invasiva que permita evaluar el bienestar animal en condiciones de estrés metabólico, ambiental y de manejo. Esta revisión ofrece una visión general de los conocimientos actuales sobre el uso potencial de miARNs tisulares y/o circulantes como biomarcadores para la evaluación del estado de salud y bienestar en el porcino