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Decoding competitive endogenous RNA regulatory network in postoperative cognitive dysfunction

Postoperative cognitive dysfunction (POCD) is a common postoperative neurological complication in elderly patients. Circular RNAs (circRNAs) are abundant in the mammalian brain and can probably regulate cognitive function. However, the competitive endogenous RNA (ceRNA) regulatory network in POCD remains illiterate. Transcriptomic signatures in the hippocampus of POCD mice derived from the Gene Expression Omnibus (GEO) dataset GSE190880, GSE95070, and GSE115440 were used to identify the circRNA, miRNA, and mRNA expression profiles of POCD mice compared with controls, respectively. A set of differentially expressed RNAs, including 119 circRNAs, 33 miRNAs, and 49 mRNAs were identified. Transcript validation showed the enhanced expression of circ_0001634, circ_0001345, and circ_0001493. A ceRNA regulatory network composed of three circRNAs, three miRNAs, and six mRNAs was established. The hub mRNAs in the ceRNA network were further found to be involved in the hormone catabolic process and regulation of canonical Wnt signaling pathway, revealing their crucial role in POCD. Finally, three miRNAs and four mRNAs were verified by qRT-PCR. These results based on bioinformatics and PCR array suggest that circ_0001634/miR-490-5p/Rbm47, circ_0001634/miR-490-5p/Sostdc1, circ_0001634/miR-7001-5p/Sostdc1, circ_0001345/miR-7001-5p/Sostdc1, and circ_0001493/miR-7001-5p/Sostdc1 may be novel diagnostic biomarkers and therapeutic targets for POCD.


Introduction
Postoperative cognitive dysfunction (POCD) is a neurocognitive disorder such as acute or persistent impairments in attention, learning, memory, and information processing that occurs predominantly in geriatric patients who undergo anesthesia and major surgery (Suwanabol et al., 2022). The long-term impact of POCD is associated with a worse overall quality of life, increased mortality, and a heavy burden on society and families (Boone et al., 2020;Tang et al., 2020). Recent evidence suggests that various elements, such as aberrant expression of apolipoprotein E4 genotype, blood-brain barrier (BBB) compromise, surgeryinduced neuroinflammation, microglial activation, mitophagy impairment, and iron accumulation (McDonagh et al., 2010;Danielson et al., 2020;Wu et al., 2020;Yang et al., 2020), are intimately involved in all stages of postoperative cognitive decline. Despite major advances in comprehending pathogenesis and prevention methods, the overall curative effect remains inadequate. Consequently, it is imperative to screen promising therapeutic targets for POCD.
Circular RNA (circRNA), unlike conventional linear RNA, is a subtype of endogenous non-coding RNAs with covalently closed-loop structures that confer high resistance to degradation (Chen, 2020). CircRNAs have been proposed to modulate gene transcription, to act as microRNA (miRNA) or RNAbinding protein (RBP) decoys, and to function as protein scaffolds (Kristensen et al., 2019). However, the overwhelming majority of circRNAs are thought to act as competing endogenous RNAs (ceRNAs), which specify that circRNAs can compete with mRNAs for binding to the shared miRNAs and thereby indirectly modify gene expression at the posttranscriptional level (Chen, 2020). The abundance of circRNAs in the mammalian brain, particularly synaptoneurosomes, is highlighted by RNA sequencing data (Rybak-Wolf et al., 2015). Mounting evidence also suggests circRNAs play a vital role in cognitive impairments such as Alzheimer impisease (AD) (Dube et al., 2019). Downregulation of circCwc27 improved cognitive capacity in the AD mouse (Song et al., 2022). Knockdown of circTshz2-2 alleviated obesity-induced spatial memory decline via modulating BDNF/TrkB signaling pathway (Yoon et al., 2021). CircPtk2 contributed to sepsisprovoked cognitive dysfunction by serving as a sponge of miR-181c-5p to facilitate HMGB1 expression (Li et al., 2021). Niu et al. (2021) proposed that aerobic exercise mitigated vascular cognitive impairment by activating circRIMS2/miR-186/BDNF axis. Additionally, plasma circRNA-089763 is positively correlated with the occurrence of POCD (Wang et al., 2019;Zhou et al., 2020). A microarray analysis screened 210 differentially expressed circRNAs in POCD patients' serum, such as circCPNE1, circUBE3B, and circITSN1 (Gao et al., 2020). Another microarray profiling highlighted three circRNAs (circ_22058, circ_44122, and circ_22673) as key elements in POCD (Wu et al., 2021). Circ_009789-and circ_004229-associated ceRNA networks were identified to elucidate the mechanism underlying susceptibility to POCD in aged mice . CircShank3 may be involved in dexmedetomidine-mediated protection against POCD via targeting the p53 and NF-κB signaling pathways . Nevertheless, the role of circRNAs in POCD remains elusive.
MiRNAs are small (18-22 nucleotide long) noncoding RNAs that trigger post-transcriptional repression of gene expression by directly binding to the 3untranslated region (UTR) of mRNAs, profoundly governing neurodevelopment and neurodegeneration (Singh and Yadav, 2020). Multiple aberrantly expressed miRNAs, such as miR-124, miR-146a, and miR-381, were implicated in the development of POCD (Chen et al., 2019a,b;Wang et al., 2021). However, the underlying mechanisms of miRNAs in the neuropathogenesis of POCD are still unclear.
In this study, we extracted the differentially expressed circRNAs, miRNAs, and mRNAs in POCD from Gene Expression Omnibus (GEO) databases. According to the flowchart diagram (Figure 1), circRNA-miRNA pairs and miRNA-mRNA pairs were successively identified for constructing a ceRNA regulatory network. Furthermore, functional enrichment analysis and transcript validation were implemented to identify the hub genes and interpret potential regulatory mechanisms in the development of POCD. The present findings may strengthen our understanding of POCD.

Expression microarray data
The circRNA dataset (GSE190880) was retrieved from the GPL21826 platform and contained 3 pairs of hippocampus from POCD and control mice (Ran et al., 2022). The miRNA dataset (GSE95070) was retrieved from the GPL19117 platform and contained 5 pairs of hippocampus from POCD and control mice (Wei et al., 2017). Meanwhile, we also downloaded the mRNA expression profiles from GSE115440 (GPL11533) (Yang et al., 2019), including 3 pairs of hippocampus tissues from POCD and control mice, to further construct the ceRNA network about POCD. The profiles of these three microarray datasets (GSE190880, GSE95070, and GSE115440) regarding circRNAs, miRNAs, and mRNAs were listed in Table 1. In these three microarray datasets, aseptic tibia fracture in C57BL/6 male mice was applied to mimic POCD.
Frontiers in Neuroscience 02 frontiersin.org Workflow of the study design. Identification of the differentially expressed circular RNAs, microRNAs, and mRNAs Differentially expressed (DE) analysis between POCD and control hippocampus samples were performed using GEO2R (Barrett et al., 2013), an online program based on R language that can analyze any GEO series to compare two groups of data. The cut-off standard of DEcircRNAs was set to p < 0.05 and |logFC| > 1.5, the cut-off standard of DEmiRNAs was set to p < 0.05 and |logFC| > 0.5, and the cut-off standard of DEmRNAs was set to p < 0.05 and |logFC| > 1. Principal component analysis (PCA) and heatmap (linkage method: row clustering; distance measure: euclidean) were visualized using the Xiantao search tool. 1

Establishment of circular RNAs-microRNA-mRNA network
The circRNA identification (ID) of DEcircRNAs were first converted into the circRNA ID in the circBase (Glažar et al., 2014), 2 an online database that integrates thousands of public circRNAs across five species and their genomic profiles. Only genomic sequences in FASTA format of annotated circRNAs were further analyzed using the circMIR1.0 software, a forecasting instrument of circRNA-miRNA interactions based on miRanda and RNAhybrid that can visualize the binding sites of miRNA adsorption. Overlapping miRNAs between the target miRNAs and DEmiRNAs screened in the GSE95070 were considered candidate miRNAs. Moreover, miRDB  3 and Targetscan (McGeary et al., 2019) (version 8.0) 4 databases were used to predict the target mRNAs of the candidate miRNAs. The Venn diagram drawn in the Xiantao search tool was utilized to overlap the miRNAforecasted mRNAs and DEmRNAs in the GSE115440. Finally, the ceRNA network was constructed using Cytoscape (Otasek et al., 2019) (version 3.9.1), by integrating circRNA-miRNA pairs and miRNA-mRNA pairs.

Functional classifications and pathway enrichment analysis
The Gene Ontology (GO) project is an ontological annotation resource that describes gene product function based on multiple databases (Ashburner et al., 2000). Kyoto Encyclopedia of Genes and Genomes (KEGG) 5 is an integrated tool that provides a new perspective on molecular-level functions, diseases and drugs (Kanehisa et al., 2016). The DAVID Knowledgebase 6 is a bioinformatics data resource based on a single-linkage method for the agglomeration of millions of genes that can be entered into DAVID gene clusters (Huang da et al., 2009a,b). GO annotation and KEGG pathway enrichment analysis were implemented using the Xiantao search tool. A pvalue of <0.05 was set as the cut-off point.

Animals
Twelve-month-old C57BL/6 male mice weighing 25-35 g were purchased from Hubei Provincial Center for Disease Control and Prevention. Mice with ad libitum access to food and water were housed in a colony room with a temperature of 22-25 • C, a humidity of 50%, and a 12-h light/dark cycle. The animals were acclimated to the environment for 7 days before experimental manipulation. All experimental procedures were approved by the Laboratory Animal Ethics Committee in Renmin Hospital of Wuhan University (No. WDRM-20210709).

Animal model of postoperative cognitive dysfunction
The animals were randomly assigned to two groups: the POCD group (n = 6) and control group (n = 6).
Intramedullary fixation for open tibial fracture under isoflurane anesthesia has been widely used as a POCD model (Yang et al., 2019). Briefly, mice were exposed to 3% isoflurane for the anesthesia induction, followed by 1.5% isoflurane in 100% oxygen for the maintenance. After shaving and disinfecting the animal, a skin incision was made on the lateral tibia, and a 0.3-mm intramedullary fixation pin was inserted into the medullary cavity. Next, an osteotomy was performed at the middle and distal third of the bone, and the wound was closed with 5-0 Vicryl suture (Ethicon, Somerville, NJ, USA). The entire procedure from the induction to the end of surgery lasted 30 min. A warm pad was utilized to keep the body temperature around 37 • C throughout the surgery. Then, 2% lidocaine solution and 1% tetracaine hydrochloride mucilage was applied locally for postoperative analgesia twice daily until 3 days after surgery. The control group received 100% oxygen without surgery or anesthesia.

Open field test
The open field test (OFT) was employed to assess the locomotor activity of the mice 3 days after the surgery. The apparatus was an opaque plastic cube box with a side length of 45 cm. Each mouse was gently placed in the central area and left to move freely for 5 min. The total distance and time spent in the center area were recorded using SuperMaze software (XinRuan Information Technology, Shanghai, China).

Morris water maze
Spatial learning and memory was assessed using Morris water maze test 4 days after the surgery. The apparatus was a circular plastic pool of 120-cm diameter and 50-cm height surrounded by four curtains. The pool was equipped with a 6-cm-diameter hidden platform placed in the center of the fourth quadrant. Prior to the experiment, the pool was filled with opaque water at around 25 • C to a depth of about 30 cm, approximately 1 cm above the platform. Each mouse received four daily trials with a 30-min intertrial interval for four consecutive days to find the platform, and was randomly placed at one of the four quadrants. Mice that failed to find the platform within 90 s were manually directed to the platform. The escape latency (time taken to reach the platform) in the four quadrants on the same day was averaged. The platform was removed on the fifth day to allow for probe trial. During the 90-s session, the total time spent in target quadrant and the frequency of crossing over the target quadrant were recorded. The swimming trajectory of the animals were recorded automatically via a video tracking system (XinRuan Information Technology, Shanghai, China).

RNA extraction and quantitative real-time polymerase chain reaction
Firstly, five circRNAs (circ_0001634, circ_0001345, circ_0001493, circ_0000487, and circ_0001468) whose host genes may be related to cognitive function were selected for quantitative real-time polymerase chain reaction (qRT-PCR). To verify the reliability and accuracy of the predicted ceRNA network, three miRNAs (miR-6912-5p, miR-490-5p, and miR-7001-5p), and four mRNAs (Rbm47, Sostdc1, Cdh3, and Sfrp5) were selected for qRT-PCR. Total RNA was extracted from the hippocampus tissue of the control and POCD group using TRIpure reagent (ELK, Wuhan, China). For circRNAs and mRNAs, cDNA was generated from template RNA using EntiLink TM Reverse Transcriptase Kit (ELK, Wuhan, China), and analyzed with EnTurbo TM SYBR Green PCR SuperMix (ELK, Wuhan, China) using StepOne TM Real-Time PCR system (Life Technologies, Carlsbad, CA, USA). The reaction procedure was as follows: 95 • C for 1 min, followed by 40 cycles of 95 • C for 15 s, 58 • C for 20 s and 72 • C for 45 s. For miRNAs, cDNA was generated using EntiLink TM 1st Strand cDNA Synthesis Kit (ELK, Wuhan, China), and analyzed with EnTurbo TM SYBR Green PCR SuperMix (ELK, Wuhan, China) using QuantStudio 6 Flex real-time PCR system (Life Technologies, Carlsbad, CA, USA). The reaction procedure was as follows: 95 • C for 30 s, followed by 95 • C for 10 s, 58 • C for 30 s, and 72 • C for 30 s. The relative expression levels of these RNAs were calculated via the 2 − Ct method. GAPDH was used as an internal control gene for circRNA and mRNA expression, while U6 was used as an internal control gene for miRNA expression. The primers for these RNAs are shown as Table 2.

Statistical analysis
Quantitative data were presented as the mean ± standard deviation (SD) or median (range). GraphPad Prism version 9.0 (GraphPad Software Inc., San Diego, California, USA) was used for statistical analyses. The statistical significance between the two groups was determined using Student's t-test or non-parametric test. A p-value of <0.05 was considered statistically significant.

Isoflurane plus orthopedic surgery caused postoperative cognitive dysfunction in mice
The open feld test showed no statistical difference in the total distance and duration of the central area between the two groups, indicating that anesthesia/surgery did not affect the locomotor activity of the mice (Figures 2A,B). To address whether isoflurane/surgery impaired cognitive function in mice, we performed Morris water maze to assess learning and memory following orthopedic surgery. During hidden platform test, the mice undergoing anesthesia/surgery showed significantly longer escape latency on day 2, 3, and 4 than the untreated control ( Figure 2C). During the probe test, time spent in target quadrant and platform crossing times in the mice undergoing anesthesia/surgery were less than those in the control group (Figures 2D-F). These results suggested that isoflurane plus orthopedic surgery caused POCD.

Data preprocessing
The accuracy and reliability of microarray data were evaluated using PCA. By comparing the distribution patterns of all identified circRNAs ( Figure 3A) and miRNAs (Figure 4A), we could completely separate POCD samples from control hippocampus.

Circular RNA expression profiles in postoperative cognitive dysfunction
In the GSE190880 dataset, a total of 119 DEcircRNAs with thresholds of |logFC| > 1.5 and p < 0.05 were detected in the hippocampus of POCD mice, of which 103 were up-regulated and 16 were down-regulated. Additionally, 76.47% of DEcircRNAs originated from exonic regions, while 8.40% originated from intronic regions. Sense-overlapping and intergenic circRNAs accounted for 13.45 and 1.68%, respectively ( Figure 3B). Chromosomal distribution demonstrated that DEcircRNAs are highly abundant in chr1-19 and chrX, but absent in chr20 and chrY ( Figure 3C).

Validation of differentially expressed circRNAs
To validate the expression of DEcircRNAs, five circRNAs whose host genes are possibly related to cognitive function were selected for verification by qRT-PCR. The expression of mmu_circ_0001634 (circFam53b), mmu_circ_0001345 (circFbxl5), and circ_0001493 (circIqsec1) were found to be upregulated in the hippocampus of POCD mice, which was consistent with the sequencing results. There was no significant difference in the expression of mmu_circ_0000487 (circXrcc4) between the two groups. Contrary to the sequencing result, isoflurane/surgery obviously diminished mmu_circ_0001468 (circHipk2) expression (Figure 5). To unveil insights into the potential functional mechanisms of circFam53b, circFbxl5, and circIqsec1, we predicted 987 miRNAs interacting with circRNAs and the putative binding domains with the aid of circMIR1.0 software ( Figure 4B).

MicroRNA expression profiles in postoperative cognitive dysfunction
A total of 33 significant DEmiRNAs with cut-off criteria of |logFC| > 0.5 and p < 0.05 were screened in the hippocampus of POCD mice. We identified 12 overlapping DEmiRNAs by integrating GSE95070 data and predicted results, of which five were upregulated and seven were downregulated ( Figures 4B,C and Supplementary Table 2). According to the ceRNA hypothesis that circRNAs could compete with mRNAs for the same miRNAs, we predicted the mRNAs downstream of 12 miRNAs. The results reflected that 2,488 target genes of the 12 DEmiRNAs were acquired by the databases of miRDB and TargetScan. Furthermore, a total of 49 significant DEmRNAs were extracted in GSE115440. The Venn diagram revealed that seven mRNAs were shared by the predicted genes of the abovementioned circRNA-targeted miRNAs and the DEmRNAs from GSE115440 (Figure 4D and Supplementary Table 3).

Construction of the competitive endogenous RNA regulatory network and experimental verification
A ceRNA regulatory network based on 3 circRNAs, 12 miRNAs, and 7 mRNAs was created to further investigate the mechanisms by which circRNAs and miRNAs affect the occurrence and development of POCD. Eventually, 10 circRNA-miRNA pairs and 7 miRNA-mRNA pairs were identified, which were composed of three circRNAs, three miRNAs, and six mRNAs ( Figure 6A). The enriched GO terms were chiefly associated with the hormone catabolic process, regulation of canonical Wnt signaling pathway, sperm midpiece, catenin complex, dipeptidyl-peptidase activity, and chloride ion binding. Moreover, the enriched KEGG pathway were renin-angiotensin system and renin secretion ( Figure 6B and Supplementary Table 4). To substantiate the potential interaction in the ceRNA, these three miRNAs and Wnt-related genes were selected for verification by qRT-PCR. The enhanced expression of miR-6912-5p, Rbm47 and Sostdc1 were observed in the POCD group, while the expression of miR-490-5p, miR-7001-5p, Cdh3, and Sfrp5 were significantly decreased in the POCD group (Figures 6C,D).

Discussion
In our study, we outlined comprehensive transcriptome profiles of POCD. A total of 119 circRNAs, 33 miRNAs, and 49 mRNAs were identified as differentially expressed. The circRNA-miRNA-mRNA triple regulatory network consisted of three circRNAs, three miRNAs, and six mRNAs. Biological process enrichment analysis revealed that the bulk of DEmRNAs in the ceRNA network were involved in the Transcript verification of circRNAs whose host genes are likely to be associated with cognitive function. Target cirRNA expression were normalized to GAPDH expression ( Ct). Non-parametric test, **p < 0.01 compared to the control group.
The vast majority of circRNAs are derived from a single or multiple exons of known coding genes and thereby located in the cytoplasm (Kristensen et al., 2019). Our analysis showed that up to 76.47% DEcircRNAs were from exons, and 14 out of 16 annotated circRNAs were of exon origin, which implied that circRNAs probably function as competing endogenous RNAs in the pathogenesis of POCD because the ceRNA network merely exists in the cytoplasm (Chen, 2020). Enrichment analysis showed the corresponding parental or host genes of DEcircRNAs were implicated in synaptic plasticity and mainly located in the Schaffer collateral-CA1 synapse, suggesting circRNAs are extensively involved in the regulation of cognitive function.
Hence, these five circRNAs were selected for verification. Quantitative RT-PCR array revealed that the augmented levels of circ_0001634, mmu_circ_0001345, and circ_0001493 were consistent with the sequencing results.
Nonetheless, some limitations of this study must be considered. First, these three datasets are retrieved from different platform and samples, which certainly reduces confidence in the regulator-target pairs outlined. Second, the ideal scenario would be the same logFC threshold and adjusted p-value. However, adjusted p-value is either absent (GSE190880) or greater than 0.9 (GSE95070 and GSE115440), which may be attributed to the relative small sample size and unknown distribution of their genomic data. There are many DEcircRNAs but few DEmiRNAs in the case of p < 0.05 and |logFC| o > 1.0. Hence, p-value and different logFC thresholds were employed for screening DERNAs, as previously described . Third, limited RNA ID information of other datasets available such as GSE165798 (Wu et al., 2021) hampers the integration of multiple datasets. Finally, the circRNA-miRNA and miRNA-mRNA interaction relationships in the ceRNA network were both based upon a prediction algorithm that required further experimental verification.
In summary, a ceRNA regulatory network in the hippocampus of POCD mice included three circRNAs, three miRNAs, and six mRNAs. Through comprehensive bioinformatic analysis, the present study broadens our horizon on the occurrence and progression of POCD.

Data availability statement
The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.

Ethics statement
The animal study was reviewed and approved by the Laboratory Animal Ethics Committee in Renmin Hospital of Wuhan University.