Exp Mol Med.  2013 Mar;45(3):e13.

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

• Han H
• Wang YH
• Qu GJ
• Sun TT
• Li FQ
• Jiang W
• Luo SS

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.

Keyword

apolipoprotein E knockout mice; atherosclerosis; genome microarray; miRNA microarray; SOCS pathway

MeSH Terms

Animals
Aorta/metabolism/pathology
Apolipoproteins E/*deficiency/metabolism
Atherosclerosis/genetics/pathology
Down-Regulation/genetics
Gene Expression Profiling
*Gene Expression Regulation
Gene Regulatory Networks/genetics
Immunohistochemistry
Male
Mice
Mice, Inbred C57BL
Mice, Knockout
MicroRNAs/*genetics/metabolism
Models, Biological
*Oligonucleotide Array Sequence Analysis
RNA, Messenger/genetics/metabolism
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction
Signal Transduction/*genetics
Suppressor of Cytokine Signaling Proteins/genetics/metabolism
Up-Regulation/genetics
Apolipoproteins E
MicroRNAs
RNA, Messenger
Suppressor of Cytokine Signaling Proteins