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Investigation of circulating serum microRNA-328-3p and microRNA-3135a expression as promising novel biomarkers for autism spectrum disorder

NT Popov
NT Popov
, 
DS Minchev
DS Minchev
, 
MM Naydenov
MM Naydenov
, 
IN Minkov
IN Minkov
 y 
TI Vachev
TI Vachev
   | 31 dic 2018
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Article Category: Original Article
Publicado en línea: 31 dic 2018
Páginas: 5 - 12
DOI: https://doi.org/10.2478/bjmg-2018-0026
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© 2018 Popov NT, Minchev DS, Naydenov MM, Minkov IN, Vachev TI, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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

MicroRNA-specific quantitative reverse transcription polymerase chain reaction primer sets.

MiRNAsPrimer Sequences (5’>3’)
miR-197-5p SLCTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG CCT CCC AC
miR-197-5p FACA CTC CAG CTG GGC GGG TAG AGA GGG CAG T
miR-500a-5p SLCTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG TCT ACC CC
miR-500a-5p FACA CTC CAG CTG GGT AAT CCT TGC TAC CTG G
miR-424-5p SLCTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG TTC AAA AC
miR-424-5p FACA CTC CAG CTG GGC AGC AGC AAT TCA TGT
Cel-miR-39 Spike-inUCA CCG GG GUA AAU UA
Cel-miR-39 SLCTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG CAA GCT GA
Cel-mi-39 FACA CTC CAG CTG GGT CAC CGG GTG TAA ATC
Universal RGTC GGC AAT TCA GTT GAG
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Medicine, Basic Medical Science, other
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