Investigation of post-transcriptional gene regulatory networks associated with autism spectrum disorders (ASD) by microRNA expression profiling of lymphoblastoid cell lines

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. The goal of this study was to reveal dysregulation in miRNA levels that are inversely correlated with altered levels of target genes that, in turn, may be associated with the underlying pathophysiology of ASD, and to provide a better understanding of the role of miRNAs as a post-transcriptional gene regulatory mechanism associated with ASD. Lymphoblastoid cell lines (LCLs) derived from peripheral lymphocytes of 14 male subjects were obtained from the Autism Genetic Resource Exchange (AGRE, Los Angeles, CA). The subjects included three pairs of monozygotic twins discordant for diagnosis of autism, a normal sibling for 2 of the twin pairs, two pairs of autistic and unaffected siblings, and a pair of normal monozygotic twins. Global miRNA expression profiling of these LCLs was performed using high-throughput miRNA microarray analysis. A reference design was used for microarray hybridization in this study. The sample miRNAs were coupled with Cy3, whereas the common reference miRNA was coupled with Cy5, and two-colored miRNA microarray analyses were carried out by cohybridizing an equal amount of both miRNA samples onto one slide. Selected differentially expressed miRNAs were confirmed by quantitative RT-PCR, and the putative target genes of two of the confirmed miRNAs were validated by knockdown and overexpression of the respective miRNAs.

自闭症谱系障碍（Autism spectrum disorders, ASD）是一类神经发育障碍，以互惠社会互动、语言发育与/或使用异常，以及受限兴趣和重复行为为核心特征。针对诊断或病情严重程度存在表型差异的同卵双胞胎的淋巴母细胞系中神经相关基因的差异表达分析显示，DNA甲基化、微小RNA（microRNAs, miRNAs）等表观遗传因素或参与ASD的发病过程。本研究的目标在于：揭示与靶基因水平改变呈负相关的miRNA表达失调情况——这类靶基因异常或与ASD的潜在病理生理机制密切相关——并进一步阐明miRNA作为转录后基因调控机制在ASD发生发展中的作用。本研究从自闭症遗传资源交换库（Autism Genetic Resource Exchange, AGRE，美国加利福尼亚州洛杉矶）获取了14名男性受试者外周淋巴细胞来源的淋巴母细胞系（Lymphoblastoid cell lines, LCLs）。受试者构成包括3对诊断存在自闭症差异的同卵双胞胎、2对双胞胎的1名正常同胞、2对自闭症与未患病同胞，以及1对正常同卵双胞胎。采用高通量miRNA微阵列分析对上述LCLs开展全基因组miRNA表达谱检测。本研究的微阵列杂交采用参考设计方案：待测miRNA标记Cy3荧光基团，通用参考miRNA标记Cy5荧光基团，将等量的两种miRNA样品共杂交至同一张芯片上，以此完成双色miRNA微阵列分析。筛选得到的差异表达miRNA经定量反转录PCR（quantitative RT-PCR, qRT-PCR）验证；针对其中两种已验证的miRNA，通过分别敲低和过表达对应miRNA，验证了其推定靶基因的表达变化。