MicroRNA signatures differentiate types, grades, and stages of breast invasive ductal carcinoma (IDC): miRNA-target interacting signaling pathways.

BACKGROUND: Invasive ductal carcinoma (IDC) is the most common form of breast cancer which accounts for 85% of all breast cancer diagnoses. Non-invasive and early stages have a better prognosis than late-stage invasive cancer that has spread to lymph nodes. The involvement of microRNAs (miRNAs) in the initiation and progression of breast cancer holds great promise for the development of molecular tools for early diagnosis and prognosis. Therefore, developing a cost effective, quick and robust early detection protocol using miRNAs for breast cancer diagnosis is an imminent need that could strengthen the health care system to tackle this disease around the world. METHODS: We have analyzed putative miRNAs signatures in 100 breast cancer samples using two independent high fidelity array systems. Unique and common miRNA signatures from both array systems were validated using stringent double-blind individual TaqMan assays and their expression pattern was confirmed with tissue microarrays and northern analysis. In silico analysis were carried out to find miRNA targets and were validated with q-PCR and immunoblotting. In addition, functional validation using antibody arrays was also carried out to confirm the oncotargets and their networking in different pathways. Similar profiling was carried out in Brca2/p53 double knock out mice models using rodent miRNA microarrays that revealed common signatures with human arrays which could be used for future in vivo functional validation. RESULTS: Expression profile revealed 85% downregulated and 15% upregulated microRNAs in the patient samples of IDC. Among them, 439 miRNAs were associated with breast cancer, out of which 107 miRNAs qualified to be potential biomarkers for the stratification of different types, grades and stages of IDC after stringent validation. Functional validation of their putative targets revealed extensive miRNA network in different oncogenic pathways thus contributing to epithelial-mesenchymal transition (EMT) and cellular plasticity. CONCLUSION: This study revealed potential biomarkers for the robust classification as well as rapid, cost effective and early detection of IDC of breast cancer. It not only confirmed the role of these miRNAs in cancer development but also revealed the oncogenic pathways involved in different progressive grades and stages thus suggesting a role in EMT and cellular plasticity during breast tumorigenesis per se and IDC in particular. Thus, our findings have provided newer insights into the miRNA signatures for the classification and early detection of IDC.

Maize Glossy2 and Glossy2-like Genes Have Overlapping and Distinct Functions in Cuticular Lipid Deposition.

Plant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a new Gl2 homolog in the maize genome, namely Gl2-like Both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence similarity to the Arabidopsis (Arabidopsis thaliana) CER2 gene. Transgenic experiments demonstrated that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential influences on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, especially at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicated that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish complementation without the need for this catalytic motif. These findings demonstrate that Gl2 and Gl2-like overlap in their cuticular lipid function, but have evolutionarily diverged to acquire nonoverlapping functions.

The impact of the GLOSSY2 and GLOSSY2-LIKE BAHD-proteins in affecting the product profile of the maize fatty acid elongase.

The maize glossy2 and glossy2-like genes are homologs, which encode proteins that belong to the BAHD family of acyltransferases. In planta genetic studies have demonstrated that these genes may be involved in the elongation of very long chain fatty acids (VLCFAs) that are precursors of the cuticular wax fraction of the plant cuticle. VLCFAs are synthesized by a fatty acyl-CoA elongase complex (FAE) that consists of four component enzymes. Previously, we functionally identified the maize FAE component enzymes by their ability to complement haploid Saccharomyces cerevisiae strains that carry lethal deletion alleles for each FAE component enzyme. In this study we used these complemented haploid strains and wild-type diploid strains to evaluate whether the co-expression of either GLOSSY2 or GLOSSY2-LIKE with individual maize FAE component enzymes affects the VLCFA product-profile of the FAE system. Wild-type diploid strains produced VLCFAs of up to 28-carbon chain length. Co-expression of GLOSSY2 or GLOSSY2-LIKE with a combination of maize 3-ketoacyl-CoA synthases stimulated the synthesis of longer VLCFAs, up to 30-carbon chain lengths. However, such results could not be recapitulated when these co-expression experiments were conducted in the yeast haploid mutant strains that lacked individual components of the endogenous FAE system. Specifically, lethal yeast mutant strains that are genetically complemented by the expression of maize FAE-component enzymes produce VLCFAs that range between 20- and 26-carbon chain lengths. However, expressing either GLOSSY2 or GLOSSY2-LIKE in these complemented strains does not enable the synthesis of longer chain VLCFAs. These results indicate that the apparent stimulatory role of GLOSSY2 or GLOSSY2-LIKE to enable the synthesis of longer chain VLCFAs in diploid yeast cells may be associated with mixing plant enzyme components with the endogenous FAE complex.

MicroRNA Signatures in Blood or Bone Marrow Distinguish Subtypes of Pediatric Acute Lymphoblastic Leukemia.

OncomiRs are microRNAs that are associated with early onset of specific cancers. To identify microRNAs involved in pediatric acute lymphoblastic leukemia (ALL) subtypes T-ALL and B-ALL, peripheral blood and bone marrow samples were independently subjected to microarray analysis using two different high-fidelity array platforms. The unique and common gene signatures from both arrays were validated by TaqMan individual assays in 100 pediatric ALL samples. Survival studies were carried out in the test set and validation set with 50 randomly selected samples in each set. MicroRNA expression profile revealed characteristic signatures for distinguishing T and B lineages and identified 51 novel microRNAs in pediatric ALL. Interestingly, the present study also revealed endogenous similarities and differences between blood and bone marrow within each ALL subtype. When Cox regression analysis was carried out with these identified microRNAs, 11 of them exhibited expression levels significantly correlated with survival. Validation of some of the common and relevant microRNAs from both arrays showed that their targets are involved in key oncogenic signaling pathways. Thus, this study suggests that microRNAs have the potential to become important diagnostic tools for identification and monitoring clinical outcomes in ALL patients.