Differentiated miRNA expression and validation of signaling pathways in apoE gene knockout mice by cross-verification microarray platform

Abstract

The microRNA (miRNA) regulation mechanisms associated with atherosclerosis are largely undocumented. Specific selection and efficient validation of miRNA regulation pathways involved in atherosclerosis development may be better assessed by contemporary microarray platforms applying cross-verification methodology. A screening platform was established using both miRNA and genomic microarrays. Microarray analysis was then simultaneously performed on pooled atherosclerotic aortic tissues from 10 Apolipoprotein E (apoE) knockout mice (apoE−/−) and 10 healthy C57BL/6 (B6) mice. Differentiated miRNAs were screened and cross-verified against an mRNA screen database to explore integrative mRNA–miRNA regulation. Gene set enrichment analysis was conducted to describe the potential pathways regulated by these mRNA–miRNA interactions. High-throughput data analysis of miRNA and genomic microarrays of knockout and healthy control mice revealed 75 differentially expressed miRNAs in apoE−/− mice at a threshold value of 2. The six miRNAs with the greatest differentiation expression were confirmed by real-time quantitative reverse-transcription PCR (qRT–PCR) in atherosclerotic tissues. Significantly enriched pathways, such as the type 2 diabetes mellitus pathway, were observed by a gene-set enrichment analysis. The enriched molecular pathways were confirmed through qRT–PCR evaluation by observing the presence of suppressor of cytokine signaling 3 (SOCS3) and SOCS3-related miRNAs, miR-30a, miR-30e and miR-19b. Cross-verified high-throughput microarrays are optimally accurate and effective screening methods for miRNA regulation profiles associated with atherosclerosis. The identified SOCS3 pathway is a potentially valuable target for future development of targeted miRNA therapies to control atherosclerosis development and progression. Mice with fatty plaques in their arteries exhibit unique expression profiles of regulatory microRNAs. If validated in humans, these microRNAs could provide a basis for the development of diagnostic biomarkers and therapeutic interventions for people with the artery-hardening disease known as atherosclerosis. Shan-Shun Luo and co-workers at the Harbin Medical University, China, compared microRNAs found in aortic tissues from healthy mice (controls) and mice lacking the apolipoprotein E gene, which develop extensive atherosclerotic lesions. They found a total of 75 differentially expressed miRNAs, 45 of which were expressed less in the apoE-deficient mice and 30 of which were expressed more in these mice. Several of these microRNAs were involved in modulating a protein called ‘suppressor of cytokine signaling 3’, a negative regulator of cytokine signaling that when overexperessed can accelerate the atherosclerotic process.