Congenital heart defects (CHD) are one of the most common defects in offspring of diabetic mothers. There is a clear association between maternal diabetes and CHD; however the underlying molecular mechanism remains unknown. We hypothesized that maternal diabetes affects with the expression of early developmental genes that regulate the essential developmental processes of the heart, thereby resulting in the pathogenesis of CHD. We analyzed genome-wide expression profiling in the developing heart of embryos from diabetic and control mice by using the oligonucleotide microarray. Microarray analysis revealed that a total of 878 genes exhibited more than 1.5 fold changes in expression level in the hearts of experimental embryos in either E13.5 or E15.5 compared with their respective controls. Expression pattern of genes that is differentially expressed in the developing heart was further examined by the real-time reverse transcriptase-polymerase chain reaction. Several genes involved in a number of molecular signaling pathways such as apoptosis, proliferation, migration and differentiation in the developing heart were differentially expressed in embryos of diabetic pregnancy. It is concluded that altered expression of several genes involved in heart development may contribute to CHD in offspring of diabetic mothers.
Overall design: Embryos with heart malformations from diabetic mice were used as the experimental samples. At least three or four embryos each from three or four different litters of diabetic or control groups were used for each microarray experiment (n = 3). Total RNA was extracted from the pooled embryonic heart tissues at E13.5 and E15.5 of diabetic and control groups using RNeasy micro kit (Qiagen, CA, USA). RNA was quantified by Nanodrop 1000 (Thermo Scientific, MA, USA) and its quality was determined using a Bioanalyzer (Agilent, CA, USA). For each sample, 100 ng of total RNA was used for the 3’ IVT Express assay according to Origen Labs SOP (Origen Labs, Singapore). The extracted total RNA was transcribed into cDNA, which was then used as a template to synthesize biotin-labeled aRNA (amplified RNA). The aRNA was then fragmented and hybridized to Affymetrix Mouse Genome 430 2.0 array(Affymetrix, CA, USA) for 16 hours at 45ºC with rotation at 60 rpm. Arrays were then washed and stained using the FS450_0004 fluidics protocol on 3 fluidics stations and scanned using an Affymetrix 3000 7G scanner (Affymetrix, CA, USA). The scanned images were inspected for hybridization efficiency. The raw data (CEL) files generated from GCOS (GeneChip Operating Software, Affymetrix, CA, USA) were imported into Expression Console (EC) 1.1 (Affymetrix, CA, USA) to perform the Mas5 normalization and generate the quality control (QC) metrics.
Less...