Study On The Role And Mechanism Of LncRNA-2870 And MiR-3587 As CeRNA Targeting HMOX1 In Renal Ischemia-reperfusion Ferroptotic Injury

Acute renal injury is a common complication in severe patients,the common cause of which is renal ischemia-reperfusion(RIR)injury,and its main pathogenesis is ferroptosis of renal tubular epithelial cells.There are few studies on the regulation of ferroptotic injury in RIR by long-chain non-coding RNA(LncRNA)and micro RNA(miRNA)through competitive endogenous RNA mechanism(ceRNA).Therefore,this study explored the ceRNA mechanism of HMOX1 regulated by LncRNA-2870 and miR-3587 through high-throughput transcriptome sequencing,bioinformatics analysis and in vitro and in vivo experiments,so as to provide a new target for the prevention and treatment of ferroptotic injury in RIR.Chapter 1 Identification of HMOX1 in renal ischemia-reperfusion injuryObjective:To identify the key molecule of ferroptosis in RIR injuryMethods:1.The datasets GSE58438 was downloaded from the Gene Expression Omnibus(GEO)database.The common differentially expressed genes(c DEGs)of control group(Ctr)compared with RIR＿3h and RIR＿24h were screened and performed enrichment analysis.2.The protein-protein interaction(PPI)network of c DEGs was constructed,and the MCODE(molecular complex detection)plugin and Cytohubba plugin of Cytoscape software were used to screen the key molecule HMOX1.3.Other RIR datasets GSE27274,GSE3219,GSE9943 were downloaded from the GEO database to verify the expression trend of the key molecule HMOX1.4.The rat renal tubular epithelial cell line NRK-52E was cultured with hypoxia/reoxygenation(HR)to establish the HR model and observe the morphological changes.5.The qRT-PCR and Western Blotting technique were used to verify the expression trend of HMOX1 in HR model.6.NRK-52E cells were treated with Erastin to explore the expression of HMOX1.Results:1.There were 80 c DEGs,including 55 up-regulated genes and 25down-regulated genes.2.Three functional clusters were created by the MCODE plugin,and the HMOX1 was determined as the key molecule by the Cytohubba plugin.3.The analysis of RIR＿120h in the datasets GSE27274,GSE3219,GSE9943and GSE58438 showed that the expression of HMOX1 was significantly increased at3-8h after reperfusion,and significantly decreased at 24h after reperfusion.4.The membrane of NRK-52E cells cultured with HR＿6h was broken and bubbled,and the nuclear size was normal.5.The expression of HMOX1 m RNA and protein in HR model decreased gradually with the reoxygenation time.6.The expression of HMOX1 m RNA and protein increased gradually with the treatment time of Erastin.Conclusion:HMOX1 is the key molecule of ferroptotic injury in RIR,which decreases with the prolongation of reoxygenation time in HR model,and the NRK-52E cells cultured with HR＿6h show ferroptotic changes.Chapter Ⅱ miR-3587 regulates renal ischemia-reperfusion ferroptotic injury by targeting HMOX1Objective:1.To predict and verify the miR-3587 targeting HMOX1.2.To clarify the regulatory role of miR-3587 in RIR ferroptotic injury.Methods:1.The online databases Target Scan,miRWalk and miRDB were used to predict the miR-3587 targeting HMOX1,the Venn diagram was drawn to obtain the intersection,and the Target Scan was used to predict the binding sequenceof miR-3587 and HMOX1 m RNA.2.Dual luciferase reporter gene assay was used to verify the targeting relationship and binding sequenceof miR-3587 and HMOX1.3.The qRT-PCR technique was used to analysis the expression trend of miR-3587 in HR model and Erastin-treated model.4.The regulation of miR-3587 inhibitor in HR model was analyzed by detection of iron ion content and mitochondrial membrane potential,transmission electron microscope,and so on.Results:1.56,48 and 11 miRNA were predicted by Target Scan,miRWalk and miRDB,respectively,and the only intersection miR-3587 of the three groups was determined through Venn diagram.The binding sequenceof miR-3587 and HMOX1 m RNA was predicted by Target Scan.2.After transfection of miR-3587 inhibitor,compared with the mutant HMOX1group,the luciferase activity of the wild type HMOX1 group was significantly increased.3.The expression of miR-3587 in HR model increased with the reoxygenation time.4.The expression of miR-3587 decreased gradually with the treatment time of Erastin.5.Compared with the HR＿6h group,the expression of HO-1(HMOX1-encoded protein)and GPx4 protein was significantly increased,the expression of ACSL4 protein was significantly decreased,the cell viability was significantly increased,the MDA content and iron ion level were significantly decreased,the mitochondrial membrane potential was significantly increased,and the structural damage of mitochondria was alleviated in the miR-3587 inhibitor group.Conclusion:miR-3587 inhibitor inhibited ferroptosis of NRK-52E cells cultured in HR＿6h by increasing HO-1.Chapter Ⅲ Construction of ceRNA network in renal ischemia-reperfusion ferroptotic injuryObjective:1.To screen the differential expression profile of LncRNA in RIR injury.2.To predict the LncRNA that can regulate HMOX1.3.To construct the LncRNA-miRNA-m RNA regulatory network of RIR injury.Methods:1.The HR＿6h model was constructed for high-throughput transcriptome sequencing.2.Using adjusted P<0.05 and|log FC|>1 as cut-off values,the differentially expressed profile of LncRNA were screened.3.The LncRNA target genes were predicted by cis-regulatory approach and performed enrichment analysis.4.The online database Coding Potential Caculator 2 was used to predict the protein-encoding ability of LncRNA-2870.5.The online database UCSC genome browser was used to predict the species conservation of LncRNA-2870.6.The qRT-PCR technique was used to analysis the expression trend of LncRNA-2870 in HR model.7.The online database Bi Bi Serv was used to predict the binding site of LncRNA-2870 and miR-3587,and the double luciferase reporter gene assay was used to verify it.8.The online database lnc Locator 2.0 was used to predict the sub-cellular localization of LncRNA-2870.9.The sub-cellular localization of LncRNA-2870,miR-3587 and HMOX1m RNA was determined by nucleocytoplasmic separation technology.Results:1.There were 1597 differentially expressed LncRNAs between the Ctr group and HR＿6h group,of which 696 were up-regulated and 901 were down-regulated,among which LncRNA-2870 could regulate HMOX1,could not encode proteins,and included a highly conserved sequence.2.The expression of LncRNA-2870 in HR model decreased with the reoxygenation time.3.LncRNA-2870 can be directly combined with miR-3587 through prediction.After transfection of miR-3587 inhibitor,compared with the mutant LncRNA-2870group,the luciferase activity of the wild type LncRNA-2870 group was significantly increased.4.In NRK-52E cells,94%of LncRNA-2870,72%of HMOX1 m RNA and 50%of miR-3587 are located in the nucleus,while 76%of the miR-3587 in the HR＿6h model is located in the nucleus.Conclusion:The differentially expressed LncRNA-2870 does not have the ability to encode proteins,and binds to miR-3587 in the nucleus to regulate the expression of HMOX1through the ceRNA mechanism.Chapter Ⅳ LncRNA-2870 regulates renal ischemia-reperfusion ferroptotic injury by ceRNA mechanismObjective:To explore the role and mechanism of LncRNA-2870 and miR-3587 as ceRNA in RIR ferroptotic injury.Methods:1.The NRK-52E cell line overexpressing/silencing LncRNA-2870,overexpressing LncRNA-2870 and silencing HMOX1 was constructed by lentiviral vector technology.2.Those technologies,including glutathione(GSH)detection,flow cytometry,transmission electron microscope and so on,were used to confirm that overexpression/silencing of LncRNA-2870 can inhibit/promote the ferroptosis of NRK-52E cells cultured at HR＿6h,that LncRNA-2870 can inhibit ferroptosis by up-regulating HMOX1,and that miR-3587 inhibitor can reverse the promoting effect of silencing LncRNA-2870 on ferroptosis.Results:1.Compared with the control group,the expression of LncRNA-2870 in the overexpression LncRNA-2870 group was significantly increased,and the expression of miR-3587 was significantly decreased,while the silencing LncRNA-2870 group was the opposite.The expression of HMOX1 m RNA in the overexpression LncRNA-2870 combined with silencing HMOX1 group was significantly decreased.2.Compared with the HR group,the cell viability and glutathione content were significantly increased;the MDA content,iron ion and ROS levels were significantly decreased;HMOX1,GPx4 m RNA and protein expressions were significantly increased,ACSL4 m RNA and protein was significantly decreased;the mitochondrial membrane potential was significantly increased,and the damage of mitochondrial structure was significantly alleviated in the overexpression LncRNA-2870 group.The silencing LncRNA-2870 group was on the contrary.3.Compared with the overexpression LncRNA-2870 group,the glutathione content was significantly decreased;the iron ion and ROS levels were significantly increased;the m RNA and protein expressions of HMOX1 and GPx4 were significantly decreased in the overexpression LncRNA-2870 combined with silencing HMOX1 group.4.Compared with the silencing LncRNA-2870 group,the glutathione content was significantly increased;the iron ion and ROS levels were significantly decreased;the HMOX1 m RNA and protein expressions were significantly increased in the silencing LncRNA-2870 combined with miR-3587 inhibitor group.Conclusion:Overexpressing/silencing LncRNA-2870 could inhibit/promote ferroptosis in NRK-52E cells cultured with HR＿6h,while silencing HMOX1 could block the inhibitory effect of overexpressing LncRNA-2870 on ferroptosis,and miR-3587inhibitor could reverse the promoting effect of silencing LncRNA-2870 on ferroptosis.Chapter Ⅴ Over-expressing LncRNA-2870 inhibits renal ischemia-reperfusion ferroptotic injuryObjective:To explore the inhibitory effect of over-expressing LncRNA-2870 on RIR ferroptotic injury.Methods:1.Lentivirus was concentrated and purified by the method of PEG-8000precipitation,and the titer of which was measured by dilution counting method.2.SD rats with over-expressing LncRNA-2870 were constructed by local injection of lentivirus in the kidney.3.The RIR injury model was constructed by the method of bilateral renal pedicle clipping for 30min and reperfusing for 24h.4.The inhibitory effect of over-expressing LncRNA-2870 on ferroptotic injury of RIR was confirmed by prussian blue staining,immunohistochemistry,transmission electron microscope and so on.Results:1.The titer of lentivirus was 1×10~8TU/m L,and 100μL was injected locally into the kidneys of rats.2.Compared with the control group,the expression of LncRNA-2870 and HMOX1 m RNA in renal tissue of over-expressing LncRNA-2870 group was significantly increased,while the expression of miR-3587 was significantly decreased.3.Compared with RIR＿24h group,renal pathological damage was significantly alleviated,serum creatinine and blood urea nitrogen were significantly decreased,NGAL content was significantly decreased,iron deposition was significantly reduced,the m RNA and protein expressions of HMOX1 and GPx4 were significantly increased,PTGS2 content was significantly decreased,the damage of mitochondrial structure was significantly alleviated in the over-expressing LncRNA-2870 group.Conclusion:Over-expressing LncRNA-2870 could inhibit RIR ferroptotic injury.