SummaryExpression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed.

Background: It is unclear whether maternal milk microRNAs are taken up by offspring.Results: Milk microRNAs are not taken up into murine offspring tissues or blood but are degraded by the digestive system.Conclusion: It is unlikely that milk microRNAs function through canonical microRNA silencing.Significance: Nutritionally derived microRNAs are unlikely to cross the intestinal barrier and influence gene expression.

The epithelial-to-mesenchymal transition (EMT) is an important mechanism for cancer progression and metastasis. Numerous in vitro and tumor-profiling studies point to the miR-200–Zeb1 axis as crucial in regulating this process, yet in vivo studies involving its regulation within a physiological context are lacking. Here, we show that miR-200 ablation in the Rip-Tag2 insulinoma mouse model induces beta-cell dedifferentiation, initiates an EMT expression program, and promotes tumor invasion. Strikingly, disrupting the miR-200 sites of the endogenous Zeb1 locus causes a similar phenotype. Reexpressing members of the miR-200 superfamily in vitro reveals that the miR-200c family and not the co-expressed and closely related miR-141 family is responsible for regulation of Zeb1 and EMT. Our results thus show that disrupting the in vivo regulation of Zeb1 by miR-200c is sufficient to drive EMT, thus highlighting the importance of this axis in tumor progression and invasion and its potential as a therapeutic target.

Charcot-Marie-Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cells.

Using GWAS in a case-control design, 7 we recently identified rs3918226 as a new hypertension susceptibility Abstract-A case-control study revealed association between hypertension and rs3918226 in the endothelial nitric oxide synthase (eNOS) gene promoter (minor/major allele, T/C allele). We aimed at substantiating these preliminary findings by target sequencing, cell experiments, and a population study. We sequenced the 140-kb genomic area encompassing the eNOS gene. In HeLa and HEK293T cells transfected with the eNOS promoter carrying either the T or the C allele, we quantified transcription by luciferase assay. In 2722 randomly recruited Europeans (53.0% women; mean age 40.1 years), we studied blood pressure change and incidence of hypertension in relation to rs3918226, using multivariable-adjusted models. Sequencing confirmed rs3918226, a binding site of E-twenty six transcription factors, as the single nucleotide polymorphism most closely associated with hypertension. In T compared with C transfected cells, eNOS promoter activity was from 20% to 40% (P<0.01) lower. In the population, systolic/diastolic blood pressure increased over 7.6 years (median) by 9.7/6.8 mm Hg in 28 TT homozygotes and by 3.8/1.9 mm Hg in 2694 C allele carriers (P≤0.0004). The blood pressure rise was 5.9 mm Hg systolic (confidence interval [CI], 0.6-11.1; P=0.028) and 4.8 mm Hg diastolic (CI, 1.5-8.2; P=0.0046) greater in TT homozygotes, with no differences between the CT and CC genotypes (P≥0.90). Among 2013 participants normotensive at baseline, 692 (34.4%) developed hypertension. The hazard ratio and attributable risk associated with TT homozygosity were 2.04 (CI, 1.24-3.37; P=0.0054) and 51.0%, respectively. In conclusion, rs3918226 in the eNOS promoter tags a hypertension susceptibility locus, TT homozygosity being associated with lesser transcription and higher risk of hypertension. Salvi et al Hypertension and eNOS 845locus. This locus lays in the promoter of the endothelial nitric oxide synthase (eNOS) gene, which encodes the enzyme that produces nitric oxide, a strong vasodilator with a key role in the regulation of systemic vascular resistance. GWAS usually points to genomic regions of interest in relation to a trait, but seldom directly identifies the causal or functional variant. In the present study, we aimed at consolidating the role of eNOS as a hypertension susceptibility gene by fine mapping the DNA sequence tagged by rs3918226, by studying the transcriptional functionality of the rs3918226 alleles in vitro, and by relating the change in BP over time to rs3918226 in a randomly recruited population sample. Methods Target SequencingFrom the HYPERGENES study, 7 we selected 44 hypertensive patients carrying ≥1 T allele and 48 healthy controls homozygous for the C allele. Analyses of the genetic data confirmed that all patients and controls were of continental Italian descent. We sequenced a 140-kb DNA region of chromosome, 7 which, in addition to eNOS, included KCNH2 mapping upstream and 6 genes mapping downstream: ATG...