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

LE Zhichen; LIU Yifan; ZHANG Wen

doi:10.14188/j.1671-8852.2024.0140

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

更新时间：2026-11-05

- miR⁃144⁃3p targets the regulation of PTEN/AKT pathway to alleviate intestinal injury in neonatal rats with necrotizing enterocolitis
- Medical Journal of Wuhan University Vol. 46, Issue 3, Pages: 283-290(2025)
- 作者机构：
  
  武汉大学中南医院小儿外科湖北武汉 430071
- 作者简介：
- 基金信息：
- DOI：10.14188/j.1671-8852.2024.0140
  CLC： R725.7
- Received：01 March 2024，
  
  Published：25 March 2025
- 稿件说明：
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乐志晨,刘一帆,张文.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[J].武汉大学学报（医学版）,2025,46(03):283-290.

LE Zhichen,LIU Yifan,ZHANG Wen.miR⁃144⁃3p targets the regulation of PTEN/AKT pathway to alleviate intestinal injury in neonatal rats with necrotizing enterocolitis[J].Medical Journal of Wuhan University,2025,46(03):283-290.
乐志晨,刘一帆,张文.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[J].武汉大学学报（医学版）,2025,46(03):283-290. DOI： 10.14188/j.1671-8852.2024.0140.

LE Zhichen,LIU Yifan,ZHANG Wen.miR⁃144⁃3p targets the regulation of PTEN/AKT pathway to alleviate intestinal injury in neonatal rats with necrotizing enterocolitis[J].Medical Journal of Wuhan University,2025,46(03):283-290. DOI： 10.14188/j.1671-8852.2024.0140.

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⁃2206 2HCl）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（

0.05），IL⁃1β、TNF⁃α、PTEN蛋白表达水平均显著升高（

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（

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（

0.05），IL⁃1β、TNF⁃α、PTEN蛋白表达水平均显著下降（

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（

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（

0.05），而AKT磷酸化水平显著下降（

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（

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。

Abstract

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 construct 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⁃2206 2HCl) 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⁃1β and TNF⁃α proteins; 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 (

0.05)

IL⁃1β

TNF⁃α

and PTEN proteins expression levels were significantly increased (

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 (

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 th

e LPS+miR⁃144⁃3p mimic group (

0.05)

IL⁃1β

TNF⁃α

and PTEN proteins expression levels were significantly decreased (

0.05)

and the intestinal histopathology score and the apoptosis percentage of intestinal epithelial cells were significantly decreased (

0.05). In the cell model

as compared with those in the LPS+miR⁃144⁃3p mimic group

miR⁃144⁃3p transcription levels and PTEN protein expression levels in the LPS+miR⁃144⁃3p mimic+AKT inhibitor group did not change significantly (

0.05)

whereas AKT phosphorylation levels were significantly decreased (

0.05)

IL⁃1β and TNF⁃α proteins expression levels and intestinal epithelial cell apoptosis ratio were all significantly elevated (

0.05).

Conclusion

The expression of miR⁃144⁃3p was significantly decreased in the intestinal tissues of NEC neonatal rats. Overexpression of miR⁃144⁃3p may attenuate intestinal injury in NEC neonatal rats by negatively regulating the expression of PTEN protein

which in turn promotes AKT phosphorylation.

关键词

Keywords

references

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