MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3’-UTRs of ENPEP, CK2-α, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-α, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40–56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-α was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-α, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-α, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.

The nitric oxide synthase family of proteins is the unique class of mammalian enzymes that metabolizes L-arginine to form nitric oxide (NO). The atherogenic action of low-density lipoproteins (LDL) m...

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.

Summary: We here describe a simple, efficient DNA sequencing procedure for hemophilia A molecular diagnosis. In severe patients we first test for the presence of factor VIII gene intron 22 inversion using a recently described single-tube PCR method. In moderate, mild, or inversion-negative severe patients we systematically sequence the promoter, all exons and splice junctions of factor VIII gene. Specially designed primers allow amplification of 23 PCR products under the same salt conditions and thermocycling parameters. The whole sequencing procedure, from blood extraction to mutation identification, can be readily done within 42 h when using regular instruments or in just 14 h when using a high-throughput sequencer. Thus, this is a versatile and cost-effective strategy with little hands-on time requirements. Since its implementation we have identified mutations in 45/46 hemophilia A patients, 14 of which are novel. Once the genetic defect has been identified, accurate genetic counseling is then easily performed.

Human epithelial tumors need to accumulate multiple genetic alterations to form invasive carcinomas. These genetic alterations are related with growth factor receptors, cell signalling, the cell cycle and cell invasiveness. Importantly, cells need to avoid senescence and become immortalized for this process. Recently, five genes: RPS6KA6, HDAC4, KIAA0828, TCP1 and Tip60, which modulate p53-dependent function and avoid senescence were identified in a large-scale RNA interference screen. Twenty colon, 20 prostate and 20 lung carcinomas were studied to investigate whether these genes might be related with human tumors. RNA was extracted from both normal and tumor tissue from each patient. Real-time RT-PCR was performed using TaqMan probes corresponding to the RPS6KA6, HDAC4, KIAA0828, TCP1, Tip60 and p53 genes. In colon carcinomas, the RPS6KA6, HDAC4, KIAA0828 and Tip60 genes were downregulated in tumor tissue as compared with normal tissue (P < 0.001 for all genes). In lung carcinomas, HDAC4, KIAA0820 and Tip60 were downregulated (P < 0.01, P < 0.001 and P < 0.001 respectively). Whereas no significant differences were observed in prostate carcinomas, striking downregulation of the RPS6KA6 and KIAA0828 genes was observed in colon carcinomas and KIAA0828 in a subset of lung carcinomas. mRNA expression of these genes may control p53 function as well as the ras-MAPK pathway, methylation and transcriptional cellular programs. These results could unravel a novel set of regulatory suppressor genes involved in human colon and lung tumors.

Molecular diagnosis of von Willebrand Disease (VWD) is particularly complex. The autosomal von Willebrand factor gene (VWF) is large and highly polymorphic, and there is a highly homologous (>96%) partial pseudogene in chromosome 22. Because of these difficulties, application of molecular study of VWD to the clinical routine has been considerably delayed. Recent advances in sequencing technology and bioinformatics could convert direct sequencing of the complete VWF into a routine diagnostic tool for VWD, which is especially desirable in types 1 and 3. This study describes a highly optimized procedure in which all the coding and intronic flanking regions of VWF are amplified under identical thermocycling parameters in a ready-to-use PCR plate format. The entire sequencing procedure, from blood extraction to mutation identification, can be done within 24 hours, resulting in a simple, versatile, cost-effective strategy with little hands-on time requirements. To validate the method, we performed full-length VWF sequencing of 21 index cases including seven of each VWD type. A total of 30 VWF genetic variations were identified. Twelve of these sequence variations are new, including four missense, one nonsense, one insertion, the first insertion-deletion described in VWF, and 5 potential splice site mutations. To our knowledge, this is the fastest and most efficient protocol designed to date for complete sequencing of the VWF coding region in the molecular diagnosis of VWD.