miR-144-3p靶向调节PTEN/AKT通路减轻坏死性小肠结肠炎新生大鼠肠损伤  

miR-144-3p targets the regulation of PTEN/AKT pathway to alleviate intestinal injury in neonatal rats with necrotizing enterocolitis

作　　者：乐志晨 刘一帆[1] 张文[1] LE Zhichen;LIU Yifan;ZHANG Wen(Dept.of Pediatric Surgery,Zhongnan Hospital of Wuhan University,Wuhan 430071,Hubei,China)

机构地区：[1]武汉大学中南医院小儿外科,湖北武汉430071

出　　处：《武汉大学学报(医学版)》2025年第3期283-290,共8页Medical Journal of Wuhan University

基　　金：武汉大学中南医院优秀博士(后)项目(编号:ZNYB2020025)。

摘　　要：目的:探讨坏死性小肠结肠炎(NEC)新生大鼠肠组织中miR-144-3p的表达,并进一步探究miR-144-3p对NEC新生大鼠肠损伤的影响及可能机制。方法:选取2日龄新生大鼠,采用高渗配方奶喂养、缺氧、冷刺激和LPS灌胃构建NEC动物模型,随机分为正常组、NEC组、NEC+agomir-NC组、NEC+miR-144-3p agomir组,每组6只。体外培养大鼠小肠隐窝上皮细胞(IEC-6),采用LPS诱导NEC肠上皮细胞模型,分为对照组、LPS组、LPS+mimic NC组、LPS+miR-144-3p mimic组、LPS+miR-144-3p mimic+AKT抑制剂(MK-22062HCl)组。双荧光素酶报告基因验证miR-144-3p与PTEN的靶向关系;qRT-PCR检测miR-144-3p转录水平;ELISA检测IL-1β和TNF-α蛋白水平;Western Blot检测PTEN、AKT、p-AKT蛋白的表达;HE染色检测肠组织病理改变;流式细胞术检测IEC-6细胞凋亡情况。结果:双荧光素酶报告基因证实PTEN是miR-144-3p的靶基因。在NEC动物和肠上皮细胞模型中,与各自空白对照组比,NEC模型组miR-144-3p转录水平、AKT磷酸化水平均显著下降(P<0.05),IL-1β、TNF-α、PTEN蛋白表达水平均显著升高(P<0.05),肠组织病理学评分和肠上皮细胞凋亡比例均显著升高(P<0.05)。与各自阴性对照组比,NEC+miR-144-3p agomir组和LPS+miR-144-3p mimic组miR-144-3p转录水平、AKT磷酸化水平均显著升高(P<0.05),IL-1β、TNF-α、PTEN蛋白表达水平均显著下降(P<0.05),肠组织病理学评分和肠上皮细胞凋亡比例均显著下降(P<0.05)。在细胞模型中,与LPS+miR-144-3p mimic组比,LPS+miR-144-3p mimic+AKT抑制剂组miR-144-3p转录水平、PTEN蛋白表达水平变化不显著(P>0.05),而AKT磷酸化水平显著下降(P<0.05),IL-1β、TNF-α蛋白表达水平和肠上皮细胞凋亡比例均显著升高(P<0.05)。结论:miR-144-3p在NEC新生大鼠肠组织中表达明显下降,过表达miR-144-3p可能通过负性调控PTEN蛋白的表达,进而促进AKT磷酸化,减轻NEC新生大鼠肠损伤。Objective:To detect the expression of miR-144-3p in the intestinal tissues of neonatal rats with necrotizing enterocolitis(NEC)and explore the effect of miR-144-3p on intestinal injury in neonatal rats with NEC and its possible mechanism.Methods:Two-day-old neonatal rats were selected to con struct the NEC animal model using hypertonic formula milk feeding,hypoxia,cold stimulation,and LPS gavage and were randomly divided into the normal group,NEC group,NEC+agomir-NC group,and NEC+miR-144-3p agomir group,with six rats in each group.Rat small intestinal epithelial cells(IEC-6)were cultured in vitro,and the NEC intestinal epithelial cell model was induced by LPS.The cells were divided into control,LPS,LPS+mimic NC,LPS+miR-144-3p mimic,and LPS+miR-144-3p mimic+AKT inhibitor(MK-22062HCl)groups.The Dual luciferase reporter gene experiment was adopted to validate the miR-144-3p targeting to PTEN;qRT-PCR was performed to detect the transcription levels of miR-144-3p;ELISA was used to detect the levels of IL-1βand TNF-αproteins;Western Blot was used to detect the expression of PTEN,AKT,and p-AKT proteins;HE staining was used to detect intestinal histopathological change;Flow cytometry was performed to detect apoptosis in IEC-6 cells.Results:The dual luciferase reporter gene experiment confirmed PTEN as a target gene of miR-144-3p.In the NEC animal and intestinal epithelial cell models,miR-144-3p transcription levels and AKT phosphorylation levels were significantly decreased in the NEC model group(P<0.05),IL-1β,TNF-α,and PTEN proteins expression levels were significantly increased(P<0.05),and intestinal histopathology scores and the percentage of intestinal epithelial cell apoptosis were significantly increased as compared with those in their respective blank control groups(P<0.05).Compared with those in the respective negative control groups,miR-144-3p transcription levels and AKT phosphorylation levels were significantly higher in the NEC+miR-144-3p agomir group and the LPS+miR-144-3p mimic group(P<0.05),IL-1β,TNF-

关 键 词：坏死性小肠结肠炎 miR-144-3p PTEN 

分 类 号：R725.7[医药卫生—儿科]