Dataset selection
The GSE58438, GSE27274, GSE3219, and GSE9934 datasets were downloaded from the Gene Expression Omnibus (GEO) database. GSE58438 was utilized for hub gene screening, whereas GSE27274, GSE3219, and GSE9934 were used to confirm hub gene expression patterns. For further details regarding these datasets, see Table 1. The GSE58438 dataset was originally used to explore the protective effects of valproic acid and dexamethasone on acute renal ischemia-reperfusion injury, while the GSE3219 dataset was originally used to explore an early biomarker of ischemic acute renal failure and nephrotoxic acute renal failure. Therefore, samples with other intervention factors in the GSE58438 and GSE3219 datasets were not included in this study. The original purpose of the GSE27274 dataset was to explore the protective effect of upregulated fibrinogen expression on renal ischemia-reperfusion injury, and the renal cortex and medulla samples were sequenced separately. In order to assess genetic changes in the whole kidney, the cortical and medulla samples from the corresponding rats were combined and analyzed. The GSE9943 dataset was originally used to explore differential gene expression in the context of the renal ischemia-reperfusion response in BN and SD rats. In light of the possible heterogeneity among rat species, BN and SD rats in this dataset were analyzed separately in this study. For an overview of the study process, see Figure 1.
Common differentially expressed gene (cDEG) identification
The GSE58438 dataset was initially normalized using the R limma package, after which DEGs were identified using the following criteria: adjusted P < 0.05 and | logFC | ≥1. DEGs that were shared between comparisons of control (Ctr) and IR samples collected at the 3 h (IR_3h) and 24 h (IR_24h) time points, the RobustRankAggreg (RRA) R package was utilized. Volcano plots were constructed using the pheatmap package.
Pathway enrichment analyses
The DAVID online tool was used to conduct GO and KEGG pathway enrichment analyses, with resultant bubble charts being constructed using the R ggplot2 package.
Protein-protein interaction (PPI) network construction, analysis, and hub gene validation
The STRING database was used to construct a PPI network incorporating identified cDEGs using the default parameters, after which the network was imported into the Cytoscape program. The top 5 hub genes were identified using the Cytohubba plugin based on the Degree, Edge Percolated Component (EPC), Density of Maximum Neighborhood Component (DMNC), and Maximal Clique Centrality (MCC) topological measurements. HMOX1 was identified as the most critical hub gene based upon the weights of these top 5 hub genes under each screening method.
The R limma package was used to normalize and process the GSE27274, GSE3219, and GSE9934 datasets. HMOX1 expression was confirmed in all three datasets and for the Ctr vs IR_120h comparison in the GSE58438 dataset.
Predictive miRNA identification
The TargetScan, miRWalk, and miRDB databases were utilized to identify putative miRNA regulators of HMOX1. Venn diagrams were used to identify miRNAs that were predicted by all three of these databases.
In vitro renal ischemia-reperfusion model establishment
Rat renal tubular epithelial NRK-52E cells (Procell, CL-0174) were cultured in high-glucose DMEM containing 1% penicillin and streptomycin (Solarbio, 11965) and 10% fetal bovine serum (FBS; Gibco, 16000-044). Cells in the logarithmic phase of growth were added to culture plates. When cells were adherent after ~12 h, plates were transferred to a hypoxic incubator (1% O2, 95% N2, and 5% CO2) for 24 h. Media was then changed, and cells were transferred to a normoxic incubator (21% O2 and 5% CO2) for 3, 6, or 9 h in order to establish a renal hypoxia-reoxygenation (HR) model of IR injury. Changes in cell morphology were assessed with an Axio Observer 3 microscope (Carl Zeiss).
qPCR
RNA was extracted from renal tubular epithelial cells following exposure to hypoxic conditions for 24 h and after 3, 6, and 9 h of reoxygenation at which time HMOX1 and miR-3587 expression levels were assessed. TriZol (RNAiso Plus, Takara, 108-95-2) was used to extract RNA from all samples, and the NanoDrop 2000 instrument was used to assess RNA concentrations and purity. The EasyScript One-Step gDNA Removal and cDNA synthesis supermix (TransS, AE311-02) were used to prepare cDNA using a StepOnePlus analyzer Real-Time PCR System. All qPCR analyses were performed using a TB Green PreMix Ex Taq TM kit (Takara, RR420A) and an Applied Biosystems 7500 Real-Time PCR instrument. Primer sequences were as follows: β-Actin, positive: 5'-CTA TGA GGG TTA CGC GCT CC-3' and reverse: 5'-ATG TCA CGC ACG ATT TCC CT-3'; HMOX1, positive: 5'-CAG AAG AGG CTA AGA CCG CC-3' and reverse: 5'-TTG GTG AGG GAAA ATG TGC CA-3'. U6 and miR-3587 primers were synthesized by RiboBio (Guangzhou, China).
Cellular treatment and transfection
miR-inhibitor-nc, miR-3587-inhibitor, and a riboFECT™ CP Transfection Kit were purchased from RiboBi. NRK-52E cells in the logarithmic phase of growth were plated in 6-well plates and subjected to the following four treatment conditions: control, HR, HR + miRNA-inhibitor-nc transfection, and HR + miR-3587-inhibitor transfection. Cells were transfected with miRNA-inhibitor-nc and miR-3587-inhibitor constructs that were prepared based on provided kit directions. Briefly, after 12 h when cells were adherent and 50% confluent, the miRNA-inhibitor-nc or miR-3587-inhibitor transfection mixtures were applied and cells were incubated for 24 h. In this experiment, the HR model system was established as above, with the appropriate transfection mixture being added when the media was changed. For an overview of this experimental protocol, see Figure 7B.
Western blotting
RIPA buffer (Applygen, C1053) was used to harvest protein from appropriately treated cells, with protein levels in these extracts being quantified with a BCA kit (Applygen, P1511). Equal amounts of protein were then separated via 10% SDS-PAGE (20 μg/lane) and transferred to PVDF membranes (Millipore, IPVH00010 Immobilon-P Transfer Membrane) with a wet transfer system using 300 mA. Blots were blocked with 5% non-fat milk for 1 h at room temperature, followed by overnight incubation with primary antibodies (anti-HMOX1, Abcam, ab68477; anti-GPX4, Abcam, ab125066) at 4°C. Following three washes with TBST, membranes were incubated with HRP-conjugated secondary antibodies (ProteinTech, sa00001-2) for 1 h at room temperature. After three additional washes in TBST, Super ECL Prime (US EVERBRIGHT, S6008) was used to detect protein bands together with a BIO-RAD imaging system. ImageJ v1.8.0 (National Institutes of Health) was used for densitometric analyses of protein bands.
Analyses of cell viability, malondialdehyde (MDA) levels, and mitochondrial membrane potential (MMP)
Following successful cellular transfection and HR model establishment as detailed above, cell viability and MDA content were respectively analyzed using a cell counting kit-8 kit (CCK-8, Abmole, M4839) and an MDA determination kit (Nanjing Jiancheng, A003-1-2). Changes in MMP were assessed with an Olympus IX71 fluorescence microscope based on the directions provided with the JC-1 MMP assay kit (Abmole, ab113850).
Statistical analysis
GraphPad Prism 8.3.0 was used for all analyses, and experiments were repeated in triplicate. Differences between groups were compared via one-way ANOVAs, with P < 0.05 as the significance threshold.