Assessment of plasma microRNAs in congenital intestinal malrotation

Abstract

Intestinal malrotation in newborns often requires urgent surgical treatment, especially in the presence of volvulus. Therefore, early‑stage diagnosis is critical. In the present study, differentially expressed plasma microRNAs (miRNAs) were screened for in patients with intestinal malrotation using high‑throughput Illumina sequencing, and validated using reverse transcription‑quantitative PCR. Receiver operating characteristic curve (ROC) analysis was conducted to evaluate their specificity, sensitivity and assess their diagnostic value for intestinal malrotation. Bioinformatics analysis was performed to investigate the functions associated with the dysregulated miRNAs. A profile consisting of 28 differentially expressed plasma miRNAs was obtained, of which nine were verified to exhibit significantly altered expression. According to a ROC analysis, four of these could represent novel early‑stage, non‑invasive biomarkers for intestinal malrotation. Bioinformatics analysis demonstrated that the differentially expressed miRNAs were predominantly involved in 'metal ion transmembrane transporter activity' and 'calcium‑dependent protein binding', which may be related to the 'endocytosis' pathway. In conclusion, significantly differentially expressed plasma miRNAs were identified in congenital intestinal malrotation and their potential roles were described. These differentially expressed miRNAs may serve as biomarkers of intestinal malrotation and improve early diagnosis for this condition.

Figure 1.

Heat map and volcano plot of significantly dysregulated miRNAs. (A) Heat map and (B) volcano plot of significantly dysregulated miRNAs from the serum of patients with IM. Red indicates upregulated genes and green downregulated genes with P<0.05. miR/miRNA, microRNA; NC, negative control; IM, intestinal malrotation; DEG, differentially expressed gene; Up, upregulated; Down, downregulated.

Figure 2.

Bioinformatics analysis of the sequencing data. (A) GO enrichment analysis of BPs, CCs and MFs. P-values and the number of genes under each term are shown on the x-axis. GO term descriptions are presented along the y-axis. (B) Bubble graph of Kyoto Encyclopedia of Genes and Genomes enrichment results. The gene ratio of each pathway is indicated on the x-axis. Number of genes involved in each pathway proportional to the size of the bubbles. The color of the bubbles represent the P-values. GO, Gene Ontology; CC, cellular component; BP, biological process; MF, molecular function.

Figure 3.

Detailed view of the ‘endocytosis’ network. Each node represents a key transporter or enzyme. The red nodes indicate miRNA with significantly expression, while the green notes represent miRNAs with no significant change in expression.

Figure 4.

Expression levels of 18 candidate miRNAs in the serum of patients with IM and matched controls. miRNA/miR, microRNA; IM, intestinal malrotation. *P<0.05; **P<0.01, ***P<0.001, vs. control.

Figure 5.

Receiver operating characteristic curves of selected dysregulated miRNAs. Expression of nine candidate miRNAs was assessed using reverse transcription-quantitative PCR. The corresponding AUCs are presented in the diagrams. miRNA/miR, microRNA; AUC, area under the curve.