Serum microRNA microarray analysis identifies miR-4429 and miR-4689 are potential diagnostic biomarkers for biliary atresia

Abstract

This study aimed to investigate pathogenesis and novel diagnostic biomarkers of biliary atresia (BA). Serum samples from infants with BA and non-BA neonatal cholestasis (NC) were collected for miRNA microarray analysis, and then differentially expressed miRNAs were screened. Differentially expressed miRNAs were validated by qRT-PCR using an independent serum samples from infants with BA and NC. Diagnostic utility of validated miRNAs was further analyzed using serum samples by receiver-operating characteristic curve analysis. Totally, 13 differentially expressed miRNAs were identified including 11 down-regulated and 2 up-regulated ones. Target genes of hsa-miR-4429 and hsa-miR-4689 were significantly involved in FoxO signaling pathway. Eight differentially expressed miRNAs were chosen for validation by qRT-PCR analysis, and four miRNAs (hsa-miR-150-3p, hsa-miR-4429, hsa-miR-4689 and hsa-miR-92a-3p) were differentially expressed. The area under the curve of hsa-miR-4429 and hsa-miR-4689 was 0.789 (sensitivity = 83.33%, specificity = 80.00%) and 0.722 (sensitivity = 66.67%, specificity = 80.00%), respectively. Differentially expressed miRNAs including hsa-miR-4429 and hsa-miR-4689 might play critical roles in BA by regulating their target genes, and these two miRNAs may have the potential to become diagnostic biomarkers.

Figure 1. Hierarchical clustering analysis for the selected differentially expressed miRNAs.

The horizontal axis represents the serum samples from infants with biliary atresia (BA) (S139, S134, S138 and S137) and non-BA neonatal cholestasis (NC) controls (S207, S206, S204 and S208). The miRNA names are shown on the left vertical axis. Colored bars indicate the range of fold changes.

Figure 2. The regulatory network for differentially expressed miRNAs.

The blue nodes represent the target genes. The yellow triangles indicate differentially expressed miRNAs with the size corresponding to degree. The blue lines show the potential regulatory relationships between miRNAs and genes.

Figure 3. Protein-protein interaction (PPI) network for the predicted target genes of differentially expressed miRNAs.

Colored bars indicate the degree of genes.

Figure 4. Validation of selected miRNAs by qRT-PCR.

Serum expression levels of hsa-miR-92a-3p, hsa-miR-3911, hsa-miR-4689, hsa-miR-3196, hsa-miR-4429, hsa-miR-150-3p, hsa-miR-642b-3p and hsa-miR-1249 were measured in 10 infants with BA and 10 NC controls.

Figure 5

The expression levels of hsa-miR-150-3p, hsa-miR-4429, hsa-miR-4689 and hsa-miR-92a-3p in an independent set of serum samples from infants with BA (n = 35) and NC controls (n = 20) (A) hsa-miR-150-3p; (B) hsa-miR-4429; (C) hsa-miR-4689; (D) hsa-miR-92a-3p).

Figure 6

Receiver operating characteristic curve analysis for BA diagnosis (A) hsa-miR-4429, (B) hsa-miR-4689; AUC: area under the curve).