Offspring sex impacts DNA methylation and gene expression in placentae from women with diabetes during pregnancy

<div><p>Aims/Hypothesis</p><p>We hypothesized that diabetes during pregnancy (DDP) alters genome-wide DNA methylation in placenta resulting in differentially methylated loci of metabolically relevant genes and downstream changes in RNA and protein expression.</p><p>Methods</p><p>We mapped genome-wide DNA methylation with the Infinium 450K Human Methylation Bead Chip in term fetal placentae from Native American and Hispanic women with DDP using a nested case-control design (n = 17 pairs). RNA expression and protein levels were assayed via RNA-Seq and Western Blot.</p><p>Results</p><p>Genome-wide DNA methylation analysis revealed 465 CpG sites with significant changes for male offspring, 247 for female offspring, and 277 for offspring of both sexes (p<0.001). Placentae from female offspring were 40% more likely to have significant gains in DNA methylation compared with placentae from male offspring exposed to DDP (p<0.001). Changes in DNA methylation corresponded to changes in RNA and protein levels for 6 genes: PIWIL3, CYBA, GSTM1, GSTM5, KCNE1 and NXN. Differential DNA methylation was detected at loci related to mitochondrial function, DNA repair, inflammation, oxidative stress.</p><p>Conclusions/Interpretation</p><p>These findings begin to explain mechanisms responsible for the increased risk for obesity and type 2 diabetes in offspring of mothers with DDP.</p></div

Protein abundance for genes with changes in DNA methylation and mRNA expression.

<p>a. PIWIL3 b. CYBA c. GSTM1 d. GSTM5 e. KCNE1 f. NXN; a–f: Protein abundance normalized to Actin or cofillin. a: <i>All</i> pairs (N = 14; 7 male pairs and 7 female pairs). b-d: Male offspring pairs (n = 7). e-f: Female offspring pairs (n = 7). * p<0.05; ** p<0.05.</p

Genes with correlating RNA-Seq and protein levels.

<p>Genes with correlating RNA-Seq and protein levels.</p

CpG sites with significant changes in DNA methylation.

<p>CpG sites with (a) <i>AVDM</i> > 10% and (b) <i>AVDM</i> > 15% and p<0.001. (a and b) Black indicates methylation increase in DDP. Gray region indicates methylation loss in DDP. (c) Genes with > 1 CpG site with significant change in DNA methylation (p<0.001).</p

Gene regulatory regions of probe sites included on the Infinium 450K Human Methylation Bead Chip.

<p>TSS = Translational Start Site; UTR: Untranslated Region. a: Reference distribution of all probe sites included on the array. b, c and d: Distribution of probe site with <i>AVDM</i> > 10% and p< 0.001. b: <i>All</i> pairs c. Male offspring pairs. d. Female offspring pairs.</p

Correlation between DNA methylation, RNA-Seq, and protein levels.

<p>Correlation between DNA methylation, RNA-Seq, and protein levels.</p

CpG sites with greatest absolute change in DNA methylation status^*.

<p>CpG sites with greatest absolute change in DNA methylation status^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190698#t002fn001" target="_blank">*</a>}.</p

Placental RNA transcripts identified by RNA-Seq with differential expression^*.

<p>Placental RNA transcripts identified by RNA-Seq with differential expression^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190698#t003fn001" target="_blank">*</a>}.</p

Maternal and infant characteristics.

<p>a) HgbA1C, b) Parity c) Gestational Age (weeks). *p<0.05. (n = 17 pairs).</p

<i>Hsa-miR-765</i> suppresses DU145 cell growth, migration, and invasion.

<p>(A) <i>Hsa-miR-765</i> mimic effectively recognizes reporter with complementary sequence of <i>hsa-miR-765</i> in DU145 cells. Fold changes of luciferase activities of the <i>hsa-miR-765</i> mimic treated cells relative to the cells treated with the negative-control mimic are presented (n = 3). Transfection reagents were used as control. (B) <i>Hsa-miR-765</i> mimic reduces DU145 cell growth. MTS assay was performed on the cells treated with <i>hsa-miR-765</i> mimic or negative-control mimic or transfection control for 4 days (n = 8). (C) <i>Hsa-miR-765</i> mimic significant reduces G0/G1 to G2/M ratio in DU145. Representative DNA histograms (n = 3) are presented. (D) <i>Hsa-miR-765</i> mimic treatment causes up-regulation of cyclin A, cyclin B, and phosphorylated-cdc2 expression in DU145 cells. Protein expression levels of cell cycle regulator proteins were determined by Western blot analyses. Two independent experiments were performed and one representative set of data was presented. (E) <i>Hsa-miR-765</i> mimic suppresses DU145 cell migration and invasion as shown in transwell migration assay (top left) and invasion assay (top right), respectively. Representative micrographs of the cells after transwell migration (top left) or invasion assay (top right) are presented. Fold changes of migration (bottom left) and invasion (bottom right) of DU145 cells with either <i>hsa-miR-765</i> mimic or negative-control mimic relative to the control cells with negative-control mimic are presented (n = 3). (F) <i>Hsa-miR-765</i> mimic significantly reduces stress fibers and filopodia formations in DU145 cells. Representative micrographs and the percentages of the cells with intense stress fibers and the filopodial cells (n = 3) are presented. Student's t-test was used for comparisons with a cutoff p value of 0.05. ** p<0.01; bar = S.D.</p