Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

Background

The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer.

Results

We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein.

Conclusions

Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.     

Residential Selection across the Life Course: Adolescent Contextual and Individual Determinants of Neighborhood Disadvantage in Mid-Adulthood

Background

Numerous cross-sectional studies have examined neighborhood effects on health. Residential selection in adulthood has been stressed as an important cause of selection bias but has received little empirical attention, particularly its determinants from the earlier life course. The present study aims to examine whether neighborhood, family, school, health behaviors and health in adolescence are related to socioeconomic disadvantage of one''s neighborhood of residence in adulthood.

Methods

Based on the prospective Northern Swedish Cohort (analytical N = 971, 90.6% retention rate), information was collected at age 16 years concerning family circumstances, school adjustment, health behaviors and mental and physical health. Neighborhood register data was linked to the cohort and used to operationalize aggregated measures of neighborhood disadvantage (ND) at age 16 and 42. Data was analyzed with linear mixed models, with ND in adulthood regressed on adolescent predictors and neighborhood of residence in adolescence as the level-2 unit.

Results

Neighborhood disadvantage in adulthood was clustered by neighborhood of residence in adolescence (ICC = 8.6%). The clustering was completely explained by ND in adolescence. Of the adolescent predictors, ND (b = .14 (95% credible interval = .07–.22)), final school marks (b = −.18 (−.26–−.10)), socioeconomic disadvantage (b = .07 (.01–.14)), and, with borderline significance, school peer problems (b = .07 (−.00–.13)), were independently related to adulthood ND in the final adjusted model. In sex-stratified analyses, the most important predictors were school marks (b = −.21 (−.32–−.09)) in women, and neighborhood of residence (ICC = 15.5%) and ND (b = .20 (.09–.31)) in men.

Conclusions

These findings show that factors from adolescence – which also may impact on adult health – could influence the neighborhood context in which one will live in adulthood. This indicates that residential selection bias in neighborhood effects on health research may have its sources in early life.     

S. haematobium as a Common Cause of Genital Morbidity in Girls: A Cross-sectional Study of Children in South Africa

Background

Schistosoma (S.) haematobium infection is a common cause of genital morbidity in adult women. Ova in the genital mucosal lining may cause lesions, bleeding, pain, discharge, and the damaged surfaces may pose a risk for HIV. In a heterogeneous schistosomiasis endemic area in South Africa, we sought to investigate if young girls had genital symptoms and if this was associated with urinary S. haematobium.

Methodology

In a cross-sectional study of 18 randomly chosen primary schools, we included 1057 schoolgirls between the age of 10 and 12 years. We interviewed assenting girls, whose parents had consented to their participation and examined three urines from each of them for schistosome ova.

Principal findings

One third of the girls reported to have a history of genital symptoms. Prior schistosomal infection was reported by 22% (226/1020), this was associated with current genital symptoms (p<0.001). In regression analysis the genital symptoms were significantly associated both with urinary schistosomiasis (p<0.001) and water contact (p<0.001).

Conclusions

Even before sexually active age, a relatively large proportion of the participating girls had similar genital symptoms to those reported for adult genital schistosomiasis previously. Anti-schistosomal treatment should be considered at a young age in order to prevent chronic genital damage and secondary infections such as HIV, sexually transmitted diseases and other super-infections.     

Inverse Regulation of EGFR/HER1 and HER2-4 in Normal and Malignant Human Breast Tissue

Cross-talk between the estrogen and the EGFR/HER signalling pathways has been suggested as a potential cause of resistance to endocrine therapy in breast cancer. Here, we determined HER1-4 receptor and neuregulin-1 (NRG1) ligand mRNA expression levels in breast cancers and corresponding normal breast tissue from patients previously characterized for plasma and tissue estrogen levels. In tumours from postmenopausal women harbouring normal HER2 gene copy numbers, we found HER2 and HER4, but HER3 levels in particular, to be elevated (2.48, 1.30 and 22.27 –fold respectively; P<0.01 for each) compared to normal tissue. Interestingly, HER3 as well as HER4 were higher among ER+ as compared to ER- tumours (P=0.004 and P=0.024, respectively). HER2 and HER3 expression levels correlated positively with ER mRNA (ESR1) expression levels (r=0.525, P=0.044; r=0.707, P=0.003, respectively). In contrast, EGFR/HER1 was downregulated in tumour compared to normal tissue (0.13-fold, P<0.001). In addition, EGFR/HER1 correlated negatively to intra-tumour (r=-0.633, P=0.001) as well as normal tissue (r=-0.556, P=0.006) and plasma estradiol levels (r=-0.625, P=0.002), suggesting an inverse regulation between estradiol and EGFR/HER1 levels. In ER+ tumours from postmenopausal women, NRG1 levels correlated positively with EGFR/HER1 (r=0.606, P=0.002) and negatively to ESR1 (r=-0.769, P=0.003) and E2 levels (r=-0.542, P=0.020). Our results indicate influence of estradiol on the expression of multiple components of the HER system in tumours not amplified for HER2, adding further support to the hypothesis that cross-talk between these systems may be of importance to breast cancer growth in vivo.     

Investigating alginate production and carbon utilization in Pseudomonas fluorescens SBW25 using mass spectrometry-based metabolic profiling

Metabolic profiling of Pseudomonas fluorescens SBW25 and various mutants derived thereof was performed to explore how the bacterium adapt to changes in carbon source and upon induction of alginate synthesis. The experiments were performed at steady-state conditions in nitrogen-limited chemostats using either fructose or glycerol as carbon source. Carbon source consumption was up-regulated in the alginate producing mutant with inactivated anti-sigma factor MucA. The mucA- mutants (also non-alginate producing mucA- control strains) had a higher dry weight yield on carbon source implying a change in carbon and energy metabolism due to the inactivation of the anti-sigma factor MucA. Both LC–MS/MS and GC–MS methods were used for quantitative metabolic profiling, and major reorganization of primary metabolite pools in both an alginate producing and a carbon source dependent manner was observed. Generally, larger changes were observed among the phosphorylated glycolytic metabolites, the pentose phosphate pathway metabolites and the nucleotide pool than among amino acids and citric acid cycle compounds. The most significant observation at the metabolite level was the significantly reduced energy charge of the mucA- mutants (both alginate producing and non-producing control strains) compared to the wild type strain. This reduction was caused more by a strong increase in the AMP pool than changes in the ATP and ADP pools. The alginate-producing mucA- mutant had a slightly increased GTP pool, while the GDP and GMP pools were strongly increased compared to non-producing mucA- strains and to the wild type. Thus, whilst changes in the adenosine phosphate nucleotide pool are attributed to the mucA inactivation, adjustments in the guanosine phosphate nucleotide pool are consequences of the GTP-dependent alginate production induced by the mucA inactivation. This metabolic profiling study provides new insight into carbon and energy metabolism of the alginate producer P. fluorescens.     

Triploid Atlantic salmon Salmo salar have a higher dietary phosphorus requirement for bone mineralization during early development

The effect of a dietary phosphorus regime in freshwater on vertebra bone mineralization was assessed in diploid and triploid Atlantic salmon, Salmo salar. Fish were fed either a low phosphorus (LP) diet containing 10.5 g kg⁻¹ total phosphorus or a normal phosphorus (NP) diet containing 17.4 g kg⁻¹ total phosphorus from ∼3 to ∼65 g (day 126) in body weight. Two further groups were fed the NP diet from ∼3 g in body weight, but were then switched to the LP diet after 38 (∼10 g in body weight) or 77 (∼30 g in body weight) days. Growth, vertebral ash content (% ash) and radiologically detectable vertebra pathologies were assessed. Triploids were initially smaller than diploids, and again on day 77, but there was no ploidy effect on days 38 or 126. Vertebral ash content increased with increasing body size and those fish fed the NP diet had higher vertebral ash content than those groups fed the LP diet during the intervening time period, but this diet effect became less apparent as fish grew, with all groups having relatively equal vertebral ash content at termination. In general, triploids had lower vertebral ash content than diploids on day 38 and this was most evident in the group fed the LP diet. On day 77, those triploids fed the LP diet during the intervening time period had lower vertebral ash content than diploids. At termination on day 126, the triploids had the same vertebral ash content as diploids, irrespective of diet. There was a ploidy × diet interaction on vertebral deformities, with triploids having higher prevalences of fish with ≥1 deformed vertebra in all dietary groups except continuous NP. In conclusion, between days 0 and 77 (3–30 g body size), triploids required more dietary phosphorus than diploids in order to maintain similar vertebral ash content. A possible link between phosphorus feeding history and phosphorus demand is also discussed.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

Transcription profiling during the cell cycle shows that a subset of Polycomb-targeted genes is upregulated during DNA replication

Genome-wide gene expression analyses of the human somatic cell cycle have indicated that the set of cycling genes differ between primary and cancer cells. By identifying genes that have cell cycle dependent expression in HaCaT human keratinocytes and comparing these with previously identified cell cycle genes, we have identified three distinct groups of cell cycle genes. First, housekeeping genes enriched for known cell cycle functions; second, cell type-specific genes enriched for HaCaT-specific functions; and third, Polycomb-regulated genes. These Polycomb-regulated genes are specifically upregulated during DNA replication, and consistent with being epigenetically silenced in other cell cycle phases, these genes have lower expression than other cell cycle genes. We also find similar patterns in foreskin fibroblasts, indicating that replication-dependent expression of Polycomb-silenced genes is a prevalent but unrecognized regulatory mechanism.