MiR-124 Negatively Regulated PARP1 To Alleviate Renal Ischemia-reperfusion Injury

Background: Renal ischemia-reperfusion injury(RIRI)is a phenomenon that the kidneys recover their blood supply after ischemia and hypoxia,but the degree of ischemic renal tissue injury is aggravated.Kidney is easily affected by ischemia and hypoxia.Therefore,patients with RIRI are at high risk of acute kidney injury(AKI).AKI is the most important factor leading to the death of patients with kidney disease,which is featured with high mortality.In addition,RIRI is also an inevitable problem in kidney transplantation,which is an important factor causing delayed graft function(DGF)after kidney transplantation.However,the occurrence of renal ischemiareperfusion injury and its specific regulatory mechanism is still not fully understood.MicroRNAs are a class of non-coding RNAs that play a regulatory role by binding to target proteins and genes.In recent years,some miRNAs have been reported to be related to renal IRI.Recent studies have found that the expression of miR-124 in renal tubular epithelial cells is reduced under hypoxia,and miR-124 can negatively regulate MMP2 to inhibit the migration and mesenchymal transformation of tubule cells and play a role in kidney protection.MiR-124 has also been shown to bind to IRE-1α in mouse renal tubular epithelial cells(TCMK-1),negatively regulating endoplasmic reticulum stress and playing a protective role in renal tubular epithelial cells.However,the mechanism of miR-124 in renal ischemia-reperfusion injury in HK-2 cells remains unclear,and further studies are needed.PARP1(Poly(ADP-Ribose)Polymerase 1)is a member of the PARP family,a group of proteins existed in most eukaryotic cells.PARP1 is abnormally expressed in the process of renal IRI,and knockdown PARP1 can improve renal ischemiareperfusion injury in animal models,and PARP1 inhibitors have also been found to reverse ischemia-reperfusion injury in rat ischemia-reperfusion model,but the specific mechanism remains to be further studied.Recently,PARP1 has been found to act as an RNA binding protein to regulate RNA expression and function,and these findings have aroused great curiosity and interest.Methods: Renal ischemia-reperfusion injury models were established in HK-2 cells and mice.The renal injury and repair of mice,as well as the expression of miR-124 and PARP1,were evaluated by HE staining,PAS staining,fluorescence in situ hybridization,immunohistochemistry,and western blot.The binding sites of miR-124 to PARP1 were predicted by computer prediction software,and the binding ability of miR-124 to PARP1-3 ’UTR was verified by dual-luciferase reporter.The inhibitors of miR-124 and PARP1 were used in HK-2 cells and mice after renal ischemia-reperfusion respectively.The renal function and structure damage were observed at the animal level,and the cell viability,apoptosis and necrosis were detected at the cell level by HE staining,PAS staining,flow cytometry,western blot,and immunohistochemistry.And the expression of inflammatory molecules IL6,TNFα and signaling pathway proteins RIP1,RIP3 and caspase8.Finally,PARP1 was knocked down in HK-2 cells for RNA-seq and IRIP-seq sequencing analysis,and the mechanism of PARP1 as RNA-binding protein in HK-2cells was analyzed by bioinformatics.Results: After ischemia reperfusion loss of renal tubular cells,the expression of miR-124 decreased while the expression of PARP1 changed in the opposite way.By binding to the site of PARP1-3 ’UTR region and through specific regulatory mechanisms,the expression of PARP1 protein was negatively correlated with miR-124.In the tubule cells with ischemia-reperfusion injury,both the overexpression of miR-124 and PARP1 inhibitors can improve the viability of tubule cells,reduce cell apoptosis and necrosis,and inhibit the inflammatory factor TNFα to block the necroptosis RIP1/RIP3 signaling pathway,thus playing a role in kidney protection.RNA-seq and IRIP-seq analysis showed that PARP1 was more likely to bind extracellular matrix-related RNA to play a role in transcription and post-transcriptional regulation in HK-2 cells.Conclusion: In renal ischemia-reperfusion injury,it was found that miR-124 negatively regulates the expression of PARP1,alleviated the degree of renal injury after ischemiareperfusion by inhibiting the TNFα/RIP1/RIP3 signaling pathway,and played a protection role of renal.PARP1 acted as an RNA-binding protein to bind the extracellular matrix-related RNA,which is a new regulatory mechanism in HK-2 cells.