MicroRNA-484 is more highly expressed in serum of early breast cancer patients compared to healthy volunteers.

BACKGROUND: Previous studies have profiled breast cancer compared to normal breast tissue and identified differentially expressed microRNAs (miRNAs). These miRNAs are then assessed in serum of breast cancer patients compared to healthy volunteers. MiRNAs in serum however do not always reflect what is in tissue and important serum miRNAs may be missed. PCR arrays were therefore performed on serum samples from breast cancer patients compared to healthy volunteers with the aim of identifying circulating miRNAs that are more highly expressed in serum from early breast cancer patients compared to controls. METHODS: Taqman low density array (TLDA) cards were used to profile serum miRNAs in a discovery cohort of serum from 39 early breast cancer patients compared to 10 healthy volunteers. The results were confirmed in a validation cohort of serum from 98 early breast cancer patients compared to 25 healthy volunteers using customized qPCR plates. RESULTS: Seventeen miRNAs were found to have significantly higher levels in breast cancer serum compared to serum of healthy volunteers in the discovery cohort. Fourteen of these miRNAs were studied in the validation cohort and serum miR-484 was found to be at a significantly higher level in breast cancer serum compared to healthy volunteers. CONCLUSION: In this study, we found that miR-484 is significantly differentially expressed in serum of early breast cancer patients compared to healthy volunteers. We did not however find any correlation between miR-484 levels with histopathological parameters of the breast cancers. With further studies, miR-484 may prove useful as an adjunct to mammography for detection of early breast cancer.

Coexpression of redox partners increases the hydrocortisone (cortisol) production efficiency in CYP11B1 expressing fission yeast Schizosaccharomyces pombe.

Cytochromes P450 play a vital role in the steroid biosynthesis pathway of the adrenal gland. An example of an essential P450 cytochrome is the steroid 11beta-hydroxylase CYP11B1, which catalyses the conversion of 11-deoxycorticol to hydrocortisone. However, despite its high biotechnological potential, this enzyme has so far been unsuccessfully employed in present-day biotechnology due to a poor expression yield and inherent protein instability. In this study, CYP11B1 was biotransformed into various strains of the yeast Schizosaccharomyces pombe, all of which also expressed the electron transfer proteins adrenodoxin and/or adrenodoxin reductase - central components of the mitochondrial P450 system - in order to maximise hydrocortisone production efficiency in our proposed model system. Site-directed mutagenesis of CYP11B1 at positions 52 and 78 was performed in order to evaluate the impact of altering the amino acids at these sites. It was found that the presence of an isoleucine at position 78 conferred the highest 11beta-hydroxylation activity of CYP11B1. Coexpression of adrenodoxin and adrenodoxin reductase appeared to further increase the 11beta-hydroxylase activity of the enzyme (3.4 fold). Adrenodoxin mutants which were found to significantly enhance enzyme efficiency in other cytochromes in previous studies were also tested in our system. It was found that, in this case, the wild type adrenodoxin was more efficient. The new fission yeast strain TH75 coexpressing the wild type Adx and AdR displays high hydrocortisone production efficiency at an average of 1mM hydrocortisone over a period of 72h, the highest value published to date for this biotransformation. Finally, our research shows that pTH2 is an ideal plasmid for the coexpression of the mitochondrial electron transfer counterparts, adrenodoxin and adrenodoxin reductase, in Schizosaccharomyces pombe, and so could serve as a convenient tool for future biotechnological applications.

Efficient conversion of 11-deoxycortisol to cortisol (hydrocortisone) by recombinant fission yeast Schizosaccharomyces pombe.

Genetically engineered microorganisms are being increasingly used for the industrial production of complicated chemical compounds such as steroids; however, there have been few reports on the use of the fission yeast Schizosaccharomyces pombe for this purpose. We previously have demonstrated that this yeast is a unique host for recombinant expression of human CYP11B2 (aldosterone synthase), and here we report the functional production of human CYP11B1 (steroid 11beta-hydroxylase) in S. pombe using our new integration vector pCAD1. In the human adrenal, the mitochondrial cytochrome P450 enzyme CYP11B1 catalyses the conversion of 11-deoxycortisol to cortisol, a key reaction in cortisol biosynthesis that in addition is of fundamental interest for the technical synthesis of glucocorticoids. We observed that the endogenous mitochondrial electron transport system detected previously by us is capable of supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. Under optimised cultivation conditions the transformed yeasts show in vivo the inducible ability to efficiently and reliably convert deoxycortisol to cortisol at an average rate of 201 microM d(-1) over a period of 72h, the highest value published to date for this biotransformation.