Department of Paediatrics, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong.
Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong.
FEBS Open Bio. 2021 Jul;11(7):1907-1920. doi: 10.1002/2211-5463.13164. Epub 2021 Jun 1.
We previously demonstrated that microRNA(miR)-223 is overexpressed in intestinal tissue of infants with necrotizing enterocolitis (NEC). The objective of the current study was to identify the target gene of miR-223 and to investigate the role of the miR-223/nuclear factor I-A (NFIA) axis in cellular functions that underpin the pathophysiology of NEC. The target gene of miR-223 was identified by in silico target prediction bioinformatics, luciferase assay, and western blotting. We investigated downstream signals of miR-223 and cellular functions by overexpressing the miRNA in Caco-2 and FHs74 cells stimulated with lipopolysaccharide or lipoteichoic acid (LTA). NFIA was identified as a target gene of miR-223. Overexpression of miR-223 significantly induced MYOM1 and inhibited NFIA and RGN in Caco-2 cells, while costimulation with LTA decreased expression of GNA11, MYLK, and PRKCZ. Expression levels of GNA11, MYLK, IL-6, and IL-8 were increased, and levels of NFIA and RGN were decreased in FHs74 cells. These potential downstream genes were significantly correlated with levels of miR-223 or NFIA in primary NEC tissues. Overexpression of miR-223 significantly increased apoptosis of Caco-2 and FHs74 cells, while proliferation of FHs74 was inhibited. These results suggest that upon binding with NFIA, miR-223 regulates functional effectors in pathways of apoptosis, cell proliferation, G protein signaling, inflammation, and smooth muscle contraction. The miR-223/NFIA axis may play an important role in the pathophysiology of NEC by enhancing inflammation and tissue damage.
我们之前的研究表明，坏死性小肠结肠炎（NEC）患儿的肠道组织中 miR-223 表达过度。本研究的目的是确定 miR-223 的靶基因，并研究 miR-223/核因子 I-A（NFIA）轴在 NEC 病理生理学基础的细胞功能中的作用。通过计算机预测生物信息学、荧光素酶报告基因检测和 Western blot 分析鉴定 miR-223 的靶基因。通过在 Caco-2 和 FHs74 细胞中转染 miRNA 并刺激其脂多糖或脂磷壁酸（LTA），研究 miR-223 的下游信号和细胞功能。NFIA 被鉴定为 miR-223 的靶基因。miR-223 的过表达在 Caco-2 细胞中显著诱导 MYOM1 并抑制 NFIA 和 RGN，而 LTA 的共刺激降低了 GNA11、MYLK 和 PRKCZ 的表达。GNA11、MYLK、IL-6 和 IL-8 的表达水平增加，而 FHs74 细胞中 NFIA 和 RGN 的表达水平降低。这些潜在的下游基因与原发性 NEC 组织中 miR-223 或 NFIA 的水平显著相关。miR-223 的过表达显著增加了 Caco-2 和 FHs74 细胞的凋亡，而 FHs74 的增殖受到抑制。这些结果表明，miR-223 与 NFIA 结合后，调节细胞凋亡、细胞增殖、G 蛋白信号、炎症和平滑肌收缩途径中的功能效应物。miR-223/NFIA 轴可能通过增强炎症和组织损伤在 NEC 的病理生理学中发挥重要作用。
