Investigation of post-transcriptional gene regulatory networks associated with autism spectrum disorders by microRNA expression profiling of lymphoblastoid cell lines.
BACKGROUND
Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by abnormalities in reciprocal social interactions and language development and/or usage, and by restricted interests and repetitive behaviors. Differential gene expression of neurologically relevant genes in lymphoblastoid cell lines from monozygotic twins discordant in diagnosis or severity of autism suggested that epigenetic factors such as DNA methylation or microRNAs (miRNAs) may be involved in ASD.


METHODS
Global miRNA expression profiling using lymphoblasts derived from these autistic twins and unaffected sibling controls was therefore performed using high-throughput miRNA microarray analysis. Selected differentially expressed miRNAs were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, and the putative target genes of two of the confirmed miRNA were validated by knockdown and overexpression of the respective miRNAs.


RESULTS
Differentially expressed miRNAs were found to target genes highly involved in neurological functions and disorders in addition to genes involved in gastrointestinal diseases, circadian rhythm signaling, as well as steroid hormone metabolism and receptor signaling. Novel network analyses of the putative target genes that were inversely expressed relative to the relevant miRNA in these same samples further revealed an association with ASD and other co-morbid disorders, including muscle and gastrointestinal diseases, as well as with biological functions implicated in ASD, such as memory and synaptic plasticity. Putative gene targets (ID3 and PLK2) of two RT-PCR-confirmed brain-specific miRNAs (hsa-miR-29b and hsa-miR-219-5p) were validated by miRNA overexpression or knockdown assays, respectively. Comparisons of these mRNA and miRNA expression levels between discordant twins and between case-control sib pairs show an inverse relationship, further suggesting that ID3 and PLK2 are in vivo targets of the respective miRNA. Interestingly, the up-regulation of miR-23a and down-regulation of miR-106b in this study reflected miRNA changes previously reported in post-mortem autistic cerebellum by Abu-Elneel et al. in 2008. This finding validates these differentially expressed miRNAs in neurological tissue from a different cohort as well as supports the use of the lymphoblasts as a surrogate to study miRNA expression in ASD.


CONCLUSIONS
Findings from this study strongly suggest that dysregulation of miRNA expression contributes to the observed alterations in gene expression and, in turn, may lead to the pathophysiological conditions underlying autism.
Sarachana
Tewarit
T
Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, 2300 Eye St NW, Washington, DC 20037, USA. tewazz@gwmail.gwu.edu.
Zhou
Rulun
R
Chen
Guang
G
Manji
Husseini K
HK
Hu
Valerie W
VW