MicroRNA-682-mediated downregulation of PTEN in intestinal epithelial cells ameliorates intestinal ischemia-reperfusion injury.

Z Liu,J Xu,C Yang,Y Xiong,D Zou,Q Yang,J Jiang,H Zhan

doi:10.1038/cddis.2016.84

Abstract

Intestinal ischemia–reperfusion (I/R) injury causes inflammation and tissue damage and contributes to high morbidity and mortality, but the underlying mechanism remains elusive and effective therapies are still lacking. We report here a critical role of the microRNA 682 (miR-682) as a key regulator and therapeutic target in intestinal I/R injury. MiR-682 was markedly induced in intestinal epithelial cells (IECs) during intestinal ischemia in mice and in the human colonic epithelial cells during hypoxia, but was undetected rapidly after intestinal reperfusion in IEC of mice. MiR-682 induction during hypoxia was modulated by hypoxia-inducible factor-1α (HIF-1α). On lentivirus-mediated miR-682 overexpression in vivo during intestinal reperfusion or miR-682 mimic transfection in vitro during hypoxia, miR-682 decreased the expression of phosphatase and tensin homolog (PTEN) and subsequently activated nuclear translocation of nuclear factor kappa B (NF-κB) p65. Consequently, NF-κB activation by miR-682-mediated PTEN downregulation prevented reactive oxygen species (ROS) induction, inflammatory reaction, mitochondrial-mediated apoptosis and IEC apoptosis. The effect of miR-682-mediated PTEN/NF-κB pathway on IECs resulted in protection against intestinal I/R injury in mice. However, NF-κB chemical inhibitor reversed miR-682-mediated decreased PTEN expression, ROS induction, inflammation and IEC apoptosis. Collectively, these results identify a novel miR-682/PTEN/NF-κBp65 signaling pathway in IEC injury induced by I/R that could be targeted for therapy.

Highlights

With real-time PCR, total RNA samples extracted from isolated IECs of mice after intestinal ischemia were used for detecting the altered miRNAs expression
While miRNAs 181b-1, 370, 379, 376b showed a decrease, miRNAs 423, 337, 682, 412, 153 were upregulated (Figure 1a). Among these miRNAs, miR-682 showed an unvarying and highest upregulation after intestinal ischemia, but was undetected rapidly following 20 min of intestinal reperfusion (Figures 1b and c). miR-682 induction during intestinal ischemia– reperfusion (I/R) injury was confirmed by Northern analysis (Figure 1d). miR-682 was previously found via the massively parallel signature sequencing technology, but little is known about its targets and function
We used phosphatase and tensin homolog (PTEN) siRNA to investigate in vitro the effect of PTEN under hypoxia and found that caspase-3 activity was reduced while nuclear shift of NF-κB p65 was elevated (Figure 6f and Supplementary Figures 4D–F). These results indicate that miR-682 lessens oxidative stress, inflammatory reaction and cell apoptosis via PTEN/ NF-κB p65 pathway

Summary

Introduction

Small intestinal epithelium normally renews every 5 days with highly differentiated cells at the tip of villi removed by apoptosis, a physiological process for eliminating unwanted or damaged cells.[12,13] The small intestines with well-featured renewal process have been believed as an excellent model to research how tissue homeostasis function through the balance of cell birth and cell death.[14,15,16] Apoptosis has been demonstrated to have an important impact in tissue injury under some pathological conditions, such as ischemia– reperfusion (I/R) induced injury in intestines, brain, myocardium and liver.[17,18] Many evidence reveals that apoptosis is a major mode of cell death caused by I/R in these tissues. Toll-like receptors signaling molecular interleukin 1 receptorassociated kinase (Irak) 1 promoted by miR-146a fortified epithelial ligand responsiveness, chemokine secretion, apoptosis and mucosal barrier disruption in experimental intestinal I/R model.[19] Inhibition of Notch signaling by chemical inhibitor or siRNA could enhance the apoptosis of IEC-6 cells under the condition of hypoxia.[20] Pharmacological blockade of protein kinase C β2 induced by intestinal I/R markedly protected small intestine against oxidative-stress-stimulated apoptosis.[21] PI3K/AKT pathway was activated to protect against intestinal I/R injury.[22] the underlying. We have identified miR-682 as a key miRNA that is markedly induced during intestinal I/R injury and targets phosphatase and tensin homolog (PTEN) to activate nuclear factor kappa B (NF-κB) p65 pathway, which in turn, reduces IEC apoptosis and prevents intestinal I/R injury

Methods

Results

Conclusion