Investigation of Circulating Serum MicroRNA-328-3p and MicroRNA-3135a Expression as Promising Novel Biomarkers for Autism Spectrum Disorder

Abstract

Circulating microRNAs (miRNAs) are emerging as promising diagnostic biomarkers for autism spectrum disorder (ASD), but their usefulness for detecting ASD remains unclear. Nowadays, development of promising biomarkers for ASD remains a challenge. Recently, dysregulation of the miRNAs expression in postmortem brain tissue, serum and peripheral blood, have been associated with ASD. Circulating miRNAs are known to be secreted by a number of different cells and can interpose delivery of information into receiver cells, thus affecting their functions. Based on this fact, it is supposed that serum miRNAs could be a novel class of biomarkers for prognosis or diagnosis of pathological disorders including ASD. In the current research, we investigated whether the expression patterns of circulating miRNAs showed dysregulation in subjects diagnosed with ASD. Expression levels of serum miR-328-3p and miR-3135a were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) method of subjects diagnosed with ASD in comparison with healthy control subjects. Our data showed that miR-328-3p and miR-3135a were substantially down-regulated in ASD patients than in those of healthy control subjects. Moreover, target gene analysis of altered serum miRNAs displayed that these molecules targeted 162 genes denoted as unique validated targets in the miRWalk database, 71 of which appear to participate in biological pathways involved in synaptic pathways and neurodegenerative condition such as Alzheimer, Huntington and Parkinson diseases. Finally, the results strongly suggested that dys-regulated serum miRNAs might be involved in molecular pathways associated with ASD and miR-328-3p and miR-3135a have the potential to be promising novel biomarkers for ASD.

Keywords: Autism spectrum disorder (ASD); MicroRNA (miRNA) expression profiling; Serum miRNAs; Stem-loop quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Figure 1

The qPCR data showing a DNA melt profile result for amplification of the specific single product in qRT-PCR analysis. Specific single products corresponding to exogeneous spike-in control cel-miR-39 (panel A); miR-328-3p (panel B) and miR-3135a in ASD and healthy control patients, were confirmed by monitoring the dissociation curve (melting curve analysis). The melting temperatures of miR-3135a amplicons were 76 ± 1 °C (panel B), whereas spiked-in cel-miR-39 control had a melting temperature of 77 ± 1 °C (panel A), and melting temperatures of miR-328-3p amplicons were 79 ± 1 °C (panel C), respectively.

Figure 2

Differential expression of serum miRNAs in ASD patients. Quantitative RT-PCR analysis of miR-3135a and miR-328-3p levels. The circulating serum miRNAs signatures were identified by miRNA-specific stem-loop qRT-PCR analysis in the ASD and control groups. Expression levels of the analyzed miRNAs were normalized to spiked-in cel-miR-39 control and expressed in relation to controls.

Figure 3

Receiver operating characteristic curve analysis using differentially expressed serum miRNAs. The ROC for miR-3135a, and miR-328-3p signature in patients with ASD was performed to evaluate the prediction accuracy of selected biomarkers. The dotted diagonal line represents random classification accuracy (AUC 0.5). The ROC curves were drawn for miR-3135a, and miR-328-3p, which yielded 0.828 and 0.858 as AUC values, respectively. Combined ROC curve describe the logistic regression (LOGREGR) of the differentially expressed miRNA members (miR-3135a and miR-328-3p). Diagnostic sensitivity of combined classifiers was 78.9% with the corresponding specificity of 88.9%. The combination of the miRNAs showed a correspondence to that using only miR-328-3p as a biomarker.

Figure 4

Fold change difference of two down-regulated serum miRNAs between the ASD and TDC groups. Data are expressed as fold change of mean 2^–ΔΔCt for each miRNA after being normalized with spike-in cel-miR-39 control.

Figure 5

The KEGG pathway enrichment analysis for the targets of the identified serum miR-3135a.

Figure 6

The KEGG pathway enrichment analysis for the targets of the identified serum miR-328-3p.