Abstract：
OBJECTIVE: Necrotizing enterocolitis (NEC) is a significant contributor to neonatal mortality. This study aimed to investigate the role of high levels of miR-375-3p in breast milk in the development of NEC and elucidate its mechanism.
METHODS: Differential expression of miR-375-3p in the intestines of breast-fed and formula-fed mice was confirmed using real-time polymerase chain reaction (RT-PCR). NEC mice models were established, and intestinal injury was assessed using HE staining. RT-PCR and Western blot were conducted to examine the expression of miR-375-3p, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β (YWHAB), as well as the inflammatory in IEC-6 cells, and intestinal tissues obtained from NEC mice and patients. Flow cytometry and cell counting kit-8 (CCK-8) were employed to elucidate the impact of miR-375-3p and YWHAB on cell apoptosis and proliferation.
RESULTS: Breastfeeding increases miR-375-3p expression in the intestines. The expression of miR-375-3p in NEC intestinal tissues exhibited a significant decrease compared to the healthy group. Additionally, the expression of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) was higher in the NEC group compared to the control group. Down-regulation of miR-375-3p inhibited IEC-6 cell proliferation, increased apoptosis, and elevated secretion of inflammatory factors. Bioinformatics revealed that YWHAB may be a target of miR-375-3p. RT-PCR and Western blot indicated a down-regulation of YWHAB expression in intestines of NEC patients and mice. Furthermore, YWHAB was found to be positively connected with miR-375-3p. Knockdown miR-375-3p down-regulated YWHAB expression in cells. Inhibition of YWHAB exhibited similar effects to miR-375-3p in IEC-6 cells. YWHAB plasmid partially reverse cellular functional impairment induced by miR-375-3p knockdown.
CONCLUSIONS: Breastfeeding elevated miR-375-3p expression in intestines in neonatal mice. MiR-375-3p leads to a decrease in apoptosis of intestinal epithelial cells, an increase in cell proliferation, and a concomitant reduction in the expression of inflammatory factors partly through targeting YWHAB.
METHODS: Differential expression of miR-375-3p in the intestines of breast-fed and formula-fed mice was confirmed using real-time polymerase chain reaction (RT-PCR). NEC mice models were established, and intestinal injury was assessed using HE staining. RT-PCR and Western blot were conducted to examine the expression of miR-375-3p, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β (YWHAB), as well as the inflammatory in IEC-6 cells, and intestinal tissues obtained from NEC mice and patients. Flow cytometry and cell counting kit-8 (CCK-8) were employed to elucidate the impact of miR-375-3p and YWHAB on cell apoptosis and proliferation.
RESULTS: Breastfeeding increases miR-375-3p expression in the intestines. The expression of miR-375-3p in NEC intestinal tissues exhibited a significant decrease compared to the healthy group. Additionally, the expression of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) was higher in the NEC group compared to the control group. Down-regulation of miR-375-3p inhibited IEC-6 cell proliferation, increased apoptosis, and elevated secretion of inflammatory factors. Bioinformatics revealed that YWHAB may be a target of miR-375-3p. RT-PCR and Western blot indicated a down-regulation of YWHAB expression in intestines of NEC patients and mice. Furthermore, YWHAB was found to be positively connected with miR-375-3p. Knockdown miR-375-3p down-regulated YWHAB expression in cells. Inhibition of YWHAB exhibited similar effects to miR-375-3p in IEC-6 cells. YWHAB plasmid partially reverse cellular functional impairment induced by miR-375-3p knockdown.
CONCLUSIONS: Breastfeeding elevated miR-375-3p expression in intestines in neonatal mice. MiR-375-3p leads to a decrease in apoptosis of intestinal epithelial cells, an increase in cell proliferation, and a concomitant reduction in the expression of inflammatory factors partly through targeting YWHAB.
摘要：
目的：坏死性小肠结肠炎（NEC）是导致新生儿死亡的重要因素。本研究旨在探讨母乳中高水平miR-375-3p在NEC发生发展中的作用及其机制。
方法：使用实时聚合酶链反应（RT-PCR）确认母乳喂养和配方喂养小鼠肠道中miR-375-3p的差异表达。建立NEC小鼠模型,肠损伤采用HE染色进行评估。RT-PCR和Westernblot检测miR-375-3p的表达,酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白β（YWHAB），以及IEC-6细胞中的炎症，和从NEC小鼠和患者获得的肠组织。采用流式细胞术和细胞计数试剂盒-8(CCK-8)阐明miR-375-3p和YWHAB对细胞凋亡和增殖的影响。
结果：母乳喂养可增加肠道中miR-375-3p的表达。与健康组相比，NEC肠组织中miR-375-3p的表达显着降低。此外,白细胞介素-6（IL-6）的表达，白细胞介素-10（IL-10），与对照组相比，NEC组的肿瘤坏死因子-α（TNF-α）更高。下调miR-375-3p抑制IEC-6细胞增殖,细胞凋亡增加，炎症因子分泌升高。生物信息学显示YWHAB可能是miR-375-3p的靶标。RT-PCR和Westernblot表明NEC患者和小鼠肠道中YWHAB的表达下调。此外，发现YWHAB与miR-375-3p呈正相关。敲低miR-375-3p下调细胞中YWHAB的表达。YWHAB的抑制在IEC-6细胞中表现出与miR-375-3p相似的作用。YWHAB质粒部分逆转miR-375-3p敲低诱导的细胞功能损伤。
结论：母乳喂养可提高新生小鼠肠道中miR-375-3p的表达。MiR-375-3p导致肠上皮细胞凋亡减少，细胞增殖的增加，同时部分通过靶向YWHAB降低炎症因子的表达。
方法：使用实时聚合酶链反应（RT-PCR）确认母乳喂养和配方喂养小鼠肠道中miR-375-3p的差异表达。建立NEC小鼠模型,肠损伤采用HE染色进行评估。RT-PCR和Westernblot检测miR-375-3p的表达,酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白β（YWHAB），以及IEC-6细胞中的炎症，和从NEC小鼠和患者获得的肠组织。采用流式细胞术和细胞计数试剂盒-8(CCK-8)阐明miR-375-3p和YWHAB对细胞凋亡和增殖的影响。
结果：母乳喂养可增加肠道中miR-375-3p的表达。与健康组相比，NEC肠组织中miR-375-3p的表达显着降低。此外,白细胞介素-6（IL-6）的表达，白细胞介素-10（IL-10），与对照组相比，NEC组的肿瘤坏死因子-α（TNF-α）更高。下调miR-375-3p抑制IEC-6细胞增殖,细胞凋亡增加，炎症因子分泌升高。生物信息学显示YWHAB可能是miR-375-3p的靶标。RT-PCR和Westernblot表明NEC患者和小鼠肠道中YWHAB的表达下调。此外，发现YWHAB与miR-375-3p呈正相关。敲低miR-375-3p下调细胞中YWHAB的表达。YWHAB的抑制在IEC-6细胞中表现出与miR-375-3p相似的作用。YWHAB质粒部分逆转miR-375-3p敲低诱导的细胞功能损伤。
结论：母乳喂养可提高新生小鼠肠道中miR-375-3p的表达。MiR-375-3p导致肠上皮细胞凋亡减少，细胞增殖的增加，同时部分通过靶向YWHAB降低炎症因子的表达。
