Dysregulated miR-223 in small bowel tissue of infants with necrotizing enterocolitis and suppression of target gene Nuclear Factor 1A
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AbstractBackground and Objectives
Necrotizing enterocolitis (NEC) is probably the most serious intestinal inflammation disease that results in high morbidity and mortality in preterm infants. In our previous micro-RNA array analysis, we profiled 80 aberrantly expressed miRNAs in NEC tissues, amongst which the level of miR-223 was significantly upregulated [Ng, Chan et al. 2015; PLoS One 10(8): e0135737]. The objectives of this study were to validate the dysregulation of miR-223 in NEC tissues by quantitative (q)PCR and to identify the direct binding target gene of miR-223.
Expression level of miR-223 was investigated in NEC tissues and surgical control tissues (Surg CTL; i.e. non-inflammatory neonatal surgical conditions) by qPCR. Potential target genes of miR-223 were selected from 6 data bases in the public domain and cross-matched with our own mRNA data base of 3472 dysregulated genes in human NEC tissues (Chan, Leung et al. 2014; Ann Surg 260: 1128-1137). The mRNA levels of selected target genes were further investigated in small bowel tissues and miR-223 mimics-transfected Caco-2 cells. The luciferase reporter assay was applied to validate the direct binding between miR-223 and its target gene in HEK293 and Caco-2 cell lines.
The expression of miR-223 was significantly increased (25.2 fold) in NEC tissues compared with Surg CTL tissues (n=10 for each group; P=0.0107). Nuclear Factor 1A (NFIA), Scavenger Receptor Class B Member 1 (SCARB1), and Intercellular Adhesion Molecule 1 (ICAM1) were selected as potential target genes. The expression of NFIA (0.32 fold) and SCARB1 (0.12 fold) were significantly decreased in NEC tissues compared with Surg CTL (n=10; P<0.001), while the expression of ICAM1 was increased (3.75 fold; P=0.0059). All these three genes exhibited significant negative correlation with miR-223 (P<0.05), which is in line with the inhibitory property of miRNA. After overexpression of miR-223 in Caco-2 cells (n = 6), expression of NFIA was significantly repressed (0.87 fold; P=0.0022), whilst SCARB1 and ICAM1 were unchanged. Further validation showed a direct binding between 3’-UTR of NFIA and miR-223. MiR-223 mimics significantly inhibited the luciferase activity of wild type NFIA 3’-UTR plasmid both in HEK293 (0.75 fold; n=4, P=0.0005) and Caco-2 (0.88 fold; n=4, P=0.0007) cell lines. However, miR-223 mimics did not change the luciferase activity of mutated NFIA 3’-UTR plasmid, indicating that the interaction between miR-223 and NFIA was direct and specific. In addition, we demonstrated by Western blot analysis that miR-223 could suppress NFIA at the protein level (0.73 fold; n=5, P=0.0032).
Our study demonstrated dysregulation of miR-223 and its target gene NFIA in NEC tissues. Their interaction might play a role in NEC pathophysiology.

Acknowledgments: This study was supported by the Health and Medical Research Fund, Food and Health Bureau, the HKSAR Government (Project no: 02130566).
Ng, P. C., K. Y. Chan, K. T. Leung, Y. H. Tam, T. P. Ma, H. S. Lam, H. M. Cheung, K. H. Lee, K. F. To and K. Li (2015). "Comparative MiRNA Expressional Profiles and Molecular Networks in Human Small Bowel Tissues of Necrotizing Enterocolitis and Spontaneous Intestinal Perforation." PLoS One 10(8): e0135737.
Chan, K. Y., K. T. Leung, Y. H. Tam, H. S. Lam, H. M. Cheung, T. P. Ma, K. H. Lee, K. F. To, K. Li and P. C. Ng (2014). "Genome-wide expression profiles of necrotizing enterocolitis versus spontaneous intestinal perforation in human intestinal tissues: dysregulation of functional pathways." Ann Surg 260(6): 1128-1137.
Acceptance Date03/10/2016
All Author(s) ListWu Yuzheng, Kathy Yuen Yee Chan, Kam Tong Leung, Karen Li, Hugh Simon Lam, Pak Cheung Ng
Name of ConferenceFrontiers of Immunology in Health & Disease
Start Date of Conference03/10/2016
End Date of Conference06/10/2016
Place of ConferenceAwaji, Japan
Country/Region of ConferenceJapan
LanguagesEnglish-United Kingdom

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