Significant associations of common genetic variants in <i>KNG1</i> (NM_001102416.2) and <i>F11</i> (NM_000128.3) with plasma FXI levels.

<p>Significant associations of common genetic variants in <i>KNG1</i> (NM_001102416.2) and <i>F11</i> (NM_000128.3) with plasma FXI levels.</p

Plot of the association between variants within the <i>F11</i> locus with plasma FXI levels.

<p>Markers represented common variants organized by genomic position. Tthe diamond-shaped marker represented the top SNP (rs56810541) with the lowest variant-plasma FXI level association p-value. The left axis shows the statistical significance expressed as -log₁₀ of the p-values and colour intensities show the level of linkage disequilibrium between all variants and the top SNP. The recombination rate in the HapMap II sample [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176301#pone.0176301.ref033" target="_blank">33</a>] for this region is measured on the right axis.</p

Plot of the collapsing method association in the <i>KNG1</i> locus with plasma FXI levels.

<p>Markers represented the mean position of low-frequency variant sets. All of the markers located above the dotted horizontal line obtained a p-value <0.05 after the collapsing method association. Diamond-shaped markers represented the five significant low-frequency variant sets with controlling FWER = 0.05. The biggest diamond-shaped marker is the top low-frequency variant set.</p

Plot of the collapsing method association in the <i>F11</i> locus.

<p>Markers represented the mean position of low-frequency variant sets. The diamond-shaped marker represented the top low-frequency variant set and markers located above the dotted horizontal line are the most significant low-frequency variant sets (p-value <0.05). None of the low-frequency variant sets were significantly associated after controlling FWER = 0.05.</p

miR-125b Acts as a Tumor Suppressor in Breast Tumorigenesis via Its Novel Direct Targets ENPEP, CK2-α, CCNJ, and MEGF9

<div><p>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.</p> </div

ENPEP, CK2-α, CCNJ, and MEGF9 proteins and their correlation with miR-125b expression in breast cancer patients.

<p>(<b>A</b>) Correlation between miR-125b expression and ENPEP. (<b>B</b>) Correlation between miR-125b expression and CK2-α. (<b>C</b>) Correlation between miR-125b expression and MEGF9. (<b>D</b>) Correlation between CK2-α expression and the number of lymph node metastases (0, negative CK2-α expression; 1, positive CK2-α expression). (<b>E</b>) Schematic representation of how miR-125b might modulate these proteins and their possible interactions. *p < 0.05.</p

The expression of ENPEP, CK2-α, CCNJ, and MEGF9 in breast tumors.

<p>(<b>A</b>) Table showing a summary of ENPEP, CK2-α, CCNJ, and MEGF9 expression by western blot in 25 breast tumors. The total percentages of patients that express/overexpress these proteins are shown. 0, lack of expression; 1, positive expression/overexpression. (<b>B</b>) Protein expression analyzed by western blot of ENPEP, CK2-α, CCNJ, and MEGF9 in four breast cancer patients is indicated. (<b>C</b>) IHC analysis of two patients overexpressing CK2-α. Note the difference in the staining of the tumor (T) in comparison with that of normal (N) breast tissue.</p

miR-125b directly targets ENPEP, CK2-α, CCNJ, and MEGF9.

<p>(<b>A</b>) Western blot analysis of MEGF9, ENPEP, CCNJ, and CK2-α (subunits α and α’) in miRVec-GFP- and miRVec-125b-transduced MCF7, MDA-MB-231, and HMEC cells. (<b>B</b>) Western blot analysis of cell cycle proteins CCNB1 and CCND1 in miRVec-GFP- and miRVec-125b-transduced MCF7, MDA-MB-231, and HMEC cells. (<b>C</b>) The relative luciferase activity of HEK293T cells transfected with miR-125b mimics, anti-125b, or scrambled control in the presence of the wild-type luciferase pCI-3’-UTR constructs of ENPEP, CK2-α, CCNJ, and MEGF9 mRNAs. HEK293T cells were transfected with Lipofectamine 2000 and the final concentration of the scramble, miR-125b mimics, and anti-125b was 30 nM. The luciferase plasmids and the pCI or pCI-3’-UTR for each indicated gene were used at a final concentration of 100 ng; the Renilla plasmid was used at 10 ng. (<b>D</b>) The relative luciferase activity of HEK293T cells transfected with miR-125b mimics or scrambled control in the presence of the luciferase constructs containing the wild-type or mutant form of 3’-UTR-ENPEP, 3’-UTR-CK2-α, 3’-UTR-CCNJ, and 3’-UTR-MEGF9 mRNAs (*p < 0.05, **p < 0.01, ***p < 0.001).</p

The miRNA array in breast cancer patients.

<p>(<b>A</b>) A total of 35 significantly dysregulated miRNAs were differentially expressed in tumor (T) and normal (N) breast tissues (pool A and pool B) (p < 0.05) (see Materials and Methods). (<b>B</b>) The miRNA signature of differentially expressed miRNAs in breast cancer patients. To establish the miRNA signature, miRNAs that were differentially expressed between the two classes of tissues were compared with a <i>t</i>-test (p < 0.01). N/T: The numbers indicate the mean fold up- or downregulation in the normal tissue with respect to the tumor tissue. The most significantly deregulated miRNA is miR-125b, which shows more than a 5-fold downregulation in tumor tissue. (<b>C</b>) qRT-PCR of miR-125b in the patients analyzed in Figure 1A. The standard deviation is indicated. ***p < 0.001.</p

Role of multimeric analysis of von Willebrand factor (VWF) in von Willebrand disease (VWD) diagnosis: Lessons from the PCM-EVW-ES Spanish project

<div><p>The multimeric analysis (MA) of plasma von Willebrand factor (VWF) evaluates structural integrity and helps in the diagnosis of von Willebrand disease (VWD). This assay is a matter of controversy, being considered by some investigators cumbersome and only slightly informative. The centralised study ‘Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES)’ has been carried out by including the phenotypic assessment and the genetic analysis by next generation sequencing (NGS) of the VWF gene (VWF). The aim of the present study was to evaluate the role of MA to the diagnosis of these patients and their potential discrepancies. Two hundred and seventy out of 480 patients centrally diagnosed with VWD had normal multimers, 168 had abnormal multimers and 42 a total absence of multimers. VWF MA was of great significance in the diagnosis of 83 patients (17.3%), it was also of help in the diagnosis achieved in 365 additional patients (76%) and was not informative in 32 cases (6.7%). With regard to discrepancies, 110 out of 480 (23%) patients centrally diagnosed with VWD presented some kind of discordance between VWF:RCo/VWF:Ag and/or VWF:CB/VWF:Ag ratios, multimeric study and/or genetic results. The VWF MA was key in the presence of novel mutations as well as in cases with phenotypic discrepancies. A comparison between the contribution of MA and VWF:CB showed a clearly higher contribution of the former in the diagnostic process. These data seem to reinforce the relevance of the VWF MA in VWD diagnosis, despite all its limitations.</p></div