Microrna profiling analysis of differences between the melanoma of young adults and older adults

<p>Abstract</p> <p>Background</p> <p>This study represents the first attempt to perform a profiling analysis of the intergenerational differences in the microRNAs (miRNAs) of primary cutaneous melanocytic neoplasms in young adult and older age groups. The data emphasize the importance of these master regulators in the transcriptional machinery of melanocytic neoplasms and suggest that differential levels of expressions of these miRs may contribute to differences in phenotypic and pathologic presentation of melanocytic neoplasms at different ages.</p> <p>Methods</p> <p>An exploratory miRNA analysis of 666 miRs by low density microRNA arrays was conducted on formalin fixed and paraffin embedded tissues (FFPE) from 10 older adults and 10 young adults including conventional melanoma and melanocytic neoplasms of uncertain biological significance. Age-matched benign melanocytic nevi were used as controls.</p> <p>Results</p> <p>Primary melanoma in patients greater than 60 years old was characterized by the increased expression of miRs regulating TLR-MyD88-NF-kappaB pathway (hsa-miR-199a), RAS/RAB22A pathway (hsa-miR-204); growth differentiation and migration (hsa-miR337), epithelial mesenchymal transition (EMT) (let-7b, hsa-miR-10b/10b*), invasion and metastasis (hsa-miR-10b/10b*), hsa-miR-30a/e*, hsa-miR-29c*; cellular matrix components (hsa-miR-29c*); invasion-cytokinesis (hsa-miR-99b*) compared to melanoma of younger patients. MiR-211 was dramatically downregulated compared to nevi controls, decreased with increasing age and was among the miRs linked to metastatic processes. Melanoma in young adult patients had increased expression of hsa-miR-449a and decreased expression of hsa-miR-146b, hsa-miR-214*. MiR-30a* in clinical stages I-II adult and pediatric melanoma could predict classification of melanoma tissue in the two extremes of age groups. Although the number of cases is small, positive lymph node status in the two age groups was characterized by the statistically significant expression of hsa-miR-30a* and hsa-miR-204 (F-test, p-value < 0.001).</p> <p>Conclusions</p> <p>Our findings, although preliminary, support the notion that the differential biology of melanoma at the extremes of age is driven, in part, by deregulation of microRNA expression and by fine tuning of miRs that are already known to regulate cell cycle, inflammation, Epithelial-Mesenchymal Transition (EMT)/stroma and more specifically genes known to be altered in melanoma. Our analysis reveals that miR expression differences create unique patterns of frequently affected biological processes that clearly distinguish old age from young age melanomas. This is a novel characterization of the miRnomes of melanocytic neoplasms at two extremes of age and identifies potential diagnostic and clinico-pathologic biomarkers that may serve as novel miR-based targeted modalities in melanoma diagnosis and treatment.</p

Changes in Gene Expression Foreshadow Diet-Induced Obesity in Genetically Identical Mice

High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity

Establishing Variations in Adiposity in Diet-Induced Obesity in Male C57BL/6J Mice

<p>Frequency distribution of body weight in 219 mice at 12 wk of age, after being fed a high-fat diet for 4 wk. (A) Regression analysis between the change in body weight and fat mass (B) and change in body weight and lean body mass (C) from 8 and 12 wk of age as determined by NMR in 112 mice. (D) Regression analysis between mouse weight at weaning and the percent change of the ratio of fat mass (FM) to lean mass (LM) per week, <i>p</i> = .0105, <i>n</i> = 220. Changes in the FM/LM ratio per wk were calculated from 8–12 wk of age or from 8–14 wk of age. (E) Average percent change of the ratio of FM/LM per week for the various litter sizes, <i>p</i> = .0406, <i>n</i> = 220 (ANOVA).</p

Comparison of the Expression of Four Genes in the Mature Adipocyte and Stromal Vascular Fraction of the Epididymal and Inguinal Fat Pads Isolated from Four Pools of RNA Derived from Tissue Isolated from Eight Mice

<p>Each pool consisted of equal amounts of RNA from two mice. All comparisons between adipocyte and stromal vascular fractions were significant (<i>p</i> < 0.01) except for ING fat SFRP5 (<i>p</i> = 0.09). EPI = epididymal; ING = Inguinal</p

Positive Correlations between SFRP5 and Adiposity

<div><p>Regression analysis between SFRP5 mRNA levels and adiposity as estimated by the ratio of FM to LM in ING, EPI, and RP fat from 112 Mice Described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020081#pgen-0020081-g001" target="_blank">Figure 1</a>B.</p><p>FM = Fat Mass; LM = Lean Mass; ING = Inguinal; EPI = Epididymal; RP = Retroperitoneal</p></div

Coordinated Gene Expression

<p>Regression analyses of SFRP5, MEST, and Naked mRNA levels in RNA isolated from high and low gainer mice in inguinal fat depots suggest that regulation of this subset of genes share a common mechanism related to the degree of adiposity. Twenty mice were present in each group.</p

Chronology of Body Weight, Food Intake, and Metabolic Efficiency of Mice with Food Intake Restricted to an Amount Equivalent to That Consumed by 80% of Experimental Mice

<p>Twenty mice were in each group. Differences in phenotype between groups are marked by an asterisk (*) at a significance level of <i>p</i> < 0.01</p

Over-Expression of Four Genes in High Gainer Mice in Two Peritoneal Fat Depots and One Subcutaneous Fat Depot Illustrates that the Mechanism Leading to Over-Expression of These Genes Occurs in All Fat Depots

<p>Statistical significance was calculated by ANOVA. Twenty mice were present in each group.</p

Stability of the Adiposity Phenotype in B6 Mice

<p>Male mice (<i>n</i> = 107) were fed a low-fat chow diet (Picolab 5053) from weaning to 8 wk of age, a high-fat diet (Research Diets D12331) from 8–14 wk, then the low-fat chow diet, restricted to 80% of the amount consumed from wk 7–8, during wk 15 and16, and finally, the high-fat diet for wk 17–22. Mice were weighed weekly except during the food restriction period when body weights were measured daily until they had stabilized under these conditions. Food intake was measured weekly starting from wk 7. At the end of wk 8, 14, 16, and 22, the body composition of each mouse was analyzed by NMR. The body weight curves of mice at the upper and lower 10% of the frequency distribution at 22 wk of age are plotted in red and blue.</p