Comparative MiRNA Expressional Profiles and Molecular Networks in Human Small Bowel Tissues of Necrotizing Enterocolitis and Spontaneous Intestinal Perforation

<div><p>Background</p><p>Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are acute intestinal conditions which could result in mortality and severe morbidity in preterm infants. Our objective was to identify dysregulated micro-RNAs (miRNAs) in small bowel tissues of NEC and SIP, and their possible roles in disease pathophysiology.</p><p>Methods</p><p>We performed differential miRNA arrays on tissues of NEC (n = 4), SIP (n = 4) and surgical-control (Surg-CTL; n = 4), and validated target miRNAs by qPCR (n = 10 each group). The association of target miRNAs with 52 dysregulated mRNAs was investigated by bioinformatics on functional and base-pair sequence algorithms, and correlation in same tissue samples.</p><p>Results</p><p>We presented the first miRNA profiles of NEC, SIP and Surg-CTL intestinal tissues in preterm infants. Of 28 validated miRNAs, 21 were significantly different between NEC or SIP and Surg-CTL. Limited overlapping in the aberrant expression of miRNAs between NEC and SIP indicated their distinct molecular mechanisms. A proposed network of dysregulated miRNA/mRNA pairs in NEC suggested interaction at bacterial receptor <i>TLR4</i> (miR-31, miR-451, miR-203, miR-4793-3p), mediated <i>via</i> key transcription factors <i>NFKB2</i> (miR-203), AP-1<i>/FOSL1</i> (miR-194-3p), <i>FOXA1</i> (miR-21-3p, miR-431 and miR-1290) and <i>HIF1A</i> (miR-31), and extended downstream to pathways of angiogenesis, arginine metabolism, cell adhesion and chemotaxis, extracellular matrix remodeling, hypoxia/oxidative stress, inflammation and muscle contraction. In contrast, upregulation of miR-451 and miR-223 in SIP suggested modulation of G-protein-mediated muscle contraction.</p><p>Conclusions</p><p>The robust response of miRNA dysregulation in NEC and SIP, and concerted involvement of specific miRNAs in the molecular networks indicated their crucial roles in mucosa integrity and disease pathophysiology.</p></div

Interacting network of miRNA/mRNA pairs in SIP.

<p>A molecular network of miRNA/mRNA regulation associated with G-protein-mediated muscle contraction is illustrated in the diagram. Interactions between genes are indicated as binding (B), group relationship (GR), miRNA binding (M), phosphorylation (+P), transformation (T) and transcriptional regulation (TR). Expression changes are shown as red and blue color circles, representing up- and down-regulation. Green, red and grey arrows between target genes represent positive, negative and unspecified interactions, respectively. Big arrows between miRNA/mRNA pairs indicate experimentally proven relationships.</p

Clinical Characteristics of NEC, SIP and Surg-CTL Infants.

<p>Results are expressed as number (%) or median (interquartile range). Standard antibiotic regimens in our neonatal unit for treatment of NEC and SIP, included: (i) vancomycin, aminoglycoside or 3rd generation cephalosporin and metronidazole, or (ii) vancomycin and meropenem, depending on the severity of intra-abdominal pathology and microbial culture/sensitivity. Bold values indicate statistical significance (<i>P</i><0.016) after the Bonferroni correction.</p><p>^{<b><i>#</i></b>} Comparison not significant after the Bonferroni correction.</p><p>Clinical Characteristics of NEC, SIP and Surg-CTL Infants.</p

Pathway traced network of miRNA/mRNA pairs in NEC.

<p>A functional network on the association of selected miRNAs and potential target mRNAs was generated by the MetaCore software and sequence-based prediction. These miRNA/mRNA pairs were regulated at opposite directions and/or exhibited significant correlation in same samples. qPCR-validated expression changes of miRNAs or mRNAs are shown as red and blue color circles, representing up- and down-regulation. Green, red and grey arrows between target genes represent positive, negative and unspecified interactions, respectively. Big arrows between miRNA/mRNA pairs indicate experimentally proven relationships. Transcription factors and functional categories, including angiogenesis, arginine metabolism, cell adhesion, chemotaxis and inflammation, ECM remodeling, hypoxia/oxidative stress, and muscle contraction are highlighted in different colors. miRNAs which exhibited significant inverse correlation with specific mRNAs are underlined.</p

miRNA and mRNA Targets in Regulatory Networks of NEC and SIP.

<p>qPCR-confirmed significant over-expression (↑) and under-expression (↓) in NEC or SIP tissues compared with Surg-CTL tissues.</p><p>*Significant inverse correlation (<i>P</i><0.05) of miRNA/mRNA pairs in same NEC or SIP specimens.</p><p>miRNA and mRNA Targets in Regulatory Networks of NEC and SIP.</p

Principal component analysis of miRNA expression profiles in NEC, SIP and Surg-CTL small bowel tissues.

<p>miRNA expression profiles of NEC (n = 4), SIP (n = 4) and Surg-CTL (n = 4) tissues were analyzed by principal component analysis using the Partek Genomics Suite. The ellipsoids represent 95% confidence intervals of NEC (red), SIP (blue) and Surg-CTL (green) clusters. Each dot represents an experiment dataset. The axes correspond to principal component 1 (PC1; x-axis), PC2 (y-axis) and PC3 (z-axis).</p

Differentially Expressed miRNAs in Small Bowel Tissues from Infants with NEC (n = 10), SIP (n = 10) and Surg-CTL (n = 10) by qPCR Assay.

<p>Bold values indicate statistical significance <i>(P</i> < 0.0167).</p><p>^{<b><i>#</i></b>} Comparison not significant after Bonferroni correction.</p><p>Differentially Expressed miRNAs in Small Bowel Tissues from Infants with NEC (n = 10), SIP (n = 10) and Surg-CTL (n = 10) by qPCR Assay.</p