Molecular Mechanism Of MiR-27a-5p Regulates Ferroptosis In Intestinal Ischemia-reperfusion Injury Via Targeting HuR/Nrf2 Signaling Pathway

Objective:This study aims to explore the regulatory role of miR-27a-5p and HuR/Nrf2 in intestinal ischemia-reperfusion(I/R)injury and their potential molecular mechanism on ferroptosis by using miRNA-mRNA regulatory network.Methods:1.Establishment of intestinal I/R injury model in vivo and in vitro,and cell transfection experimentTo investigate the association between ferroptosis and intestinal I/R injury in vitro and in vivo.Clamping the superior mesenteric artery(SMA)for 45 min followed by clip removal was used to simulate intestinal I/R injury in vivo.C57BJ/6J mice(n=40,males)were randomly divided into sham,reperfused 30 min,reperfused 60 min,reperfused 120 min and reperfused 240 min groups to determine the optimal time for intestinal I/R injury.IEC-6 cell lines were incubated in medium without fetal bovine serum,placed in hypoxic environment(1%O2,5%CO2,94%N2)for 4 h,and then complete medium was replaced and reoxygenated in normal environment(5%CO2,95%O2)for different times to establish hypoxia-reoxygenation(H/R)model in vitro.In order to investigate whether ferrostatin-1(Fer-1),a ferroptosis inhibitor,has a protective effect on intestinal I/R injury in vivo and in vitro.Fer-1(10 mg/kg)was given or not given by intraperitoneal injection at 1 h prior to intestinal I/R in vivo.And C57BJ/6J mice(n=32,males)were randomly divided into sham,sham+Fer-1(10 mg/kg),I/R and I/R+Fer-1(10 mg/kg)groups.Fer-1(2 μM)was given or not given before H/R and IEC-6 cells were divided into control,control+Fer-1(2 μM)group,H/R group and H/R+Fer-1(2 μM)group in vitro.In order to investigate the regulatory relationship and effects of miR-27a-5p and HuR in intestinal I/R injury.The siRNA knockdown plasmid for RNA-binding protein HuR and the miRNA-27a-5p inhibitor plasmid were constructed,and IEC-6 cells were divided into control group,control+control plasmid group,H/R group and H/R+si-HuR group or H/R+miRNA-27a-5p inhibitor group.2.Intestinal I/R injury,and ferroptosis related indicators and signaling pathway detectionHistopathological changes in mouse small intestine were observed by H&E staining,reduced glutathione(GSH),malondialdehyde(MDA),and ferrous ion(Fe2+)were measured in intestinal homogenates,and mitochondrial damage was observed by transmission electron microscopy.Cell viability and oxidation levels were assessed using the CCK-8 assay,the reactive oxygen(ROS)probe DCFH-DA and the lipid peroxidation probe BODIPY581/591 C11,as well as the detection of GSH,MDA and Fe2+ levels.And the expression of ferroptosis-related proteins GPX4,ACSL4 and FTH1 in small intestinal tissues and IEC-6 cells was detected by Western Blot.A dual luciferase reporter verified the binding targets of miRNA-27a-5p.Real-time quantitative(qPCR)were performed to detect the transcript levels of miRNA-27a-5p,HuR and Nrf2.And Western Blot was performed to detect the pathway protein,including HuR,Nrf2 and SLC7A11.Tyramine signaling amplified immuno-fluorescence was performed to detect the effects of miRNA27a-5p on HuR and the effect of HuR on Nrf2,respectively.Results:1.Ferroptosis is involved in intestinal I/R injury and the protective effect of ferroptosis inhibitor Fer-1 on intestinal I/R injuryIn vivo and in vitro models of intestinal I/R injury,H&E staining observed the normal structure of the intestinal mucosa was destroyed,the villi of the small intestine were ruptured and shed,and inflammatory cells infiltrated in the intestinal I/R group compared to the sham group,with the Chui’s score noted to be most severe at reperfused 60 min.In the H/R model in vitro,ROS levels were significantly high at reoxygenated 3 h compared to the control group.At the same time,the level of GSH,and the ferroptosis-related protein expression levels of GPX4 and FTH1 in vivo and in vitro were decreased during reperfusion or reoxygenation,while the contents of MDA and Fe2+,and the protein expression level of ACSL4 were increased during reperfusion or reoxygenation.These results indicate that ferroptosis is involved in intestinal I/R injury.The changes of ferroptosis index were most obvious at 60 min of reperfusion in vivo and 3 h of reoxygenation in vitro,so they were used as the time for subsequent experiments.With and without the administration of Fer-1 under I/R or H/R condition in vivo and in vitro,it was observed by transmission electron microscopy and lipid peroxidation probes that after Fer-1 administration,mitochondrial damage was reduced and the ratio of lipid peroxidation to non-peroxidation was reduced,while GSH levels were increased,and the increase in MDA and Fe2+ levels due to I/R or H/R was reversed by Fer-1.Fer-1 increased GPX4 and FTH1 protein expression levels,and decreased ACSL4 protein expression levels compared to I/R or H/R group without Fer-1 administration.These results further explain that ferroptosis aggravates intestinal I/R injury and Fer-1 alleviates intestinal I/R injury by inhibiting ferroptosis.2.Identification of miR-27a-5p target gene and its mechanism of regulating intestinal I/R injury by targeting HuR/Nrf2The qPCR results pointed to a significant up-regulation of miRNA-27a-5p in H/Rinjured IEC-6 cells.The HuR was predicted by the database as its direct binding site,while the predictions were validated using a dual luciferase reporter,confirming HuR as a direct target for miRNA-27a-5p.Compared to H/R group,miRNA-27a-5p knockdown significantly reduced the protein expression of ACSL4 and increased the protein expression of GPX4,FTH1 and SLC7A11.The lipid peroxidation fluorescence probing also showed that miRNA-27a-5p knockdown significantly reduced the elevated levels of lipid peroxidation induced by H/R.Immunofluorescence and Western Blot results indicated that knockdown of miRNA-27a-5p could further elevate HuR expression in the presence of H/R.This suggests that miRNA-27a-5p can promote ferroptosis and that inhibition of miRNA-27a-5p can reduce the occurrence of ferroptosis,which is related to its direct action on the target HuR.To investigate the potential role of HuR in intestinal I/R injury,HuR knockdown further downregulated H/R-induced decreases in GPX4,FTH1 and SLC7A11 protein expression,while ACSL4 protein expression was upregulated.Immunofluorescence double staining results indicated that H/R caused an increase in total HuR and Nrf2,and that HuR appeared to be translocated into the cytoplasm while Nrf2 was translocated into the nucleus.Whereas HuR knockdown resulted in a downregulation of not only HuR expression but also Nrf2 expression.This suggests that HuR plays a protective role in intestinal I/R injury by reducing ferroptosis,which may be achieved by regulating the expression of Nrf2.Conclusion:(1)Ferroptosis is involved in intestinal I/R damage,and miRNA-27a-5p plays an important role in this process.(2)miRNA-27a-5p promotes intestinal I/R injury by blocking HuR/Nrf2 signaling regulatory pathways.(3)HuR may be an important drug target for intestinal I/R treatment.