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Frontiers | The Potential Round RNAs Biomarker Panel and Regulatory Networks of Parkinson’s Illness


Introduction

Parkinson’s illness (PD) is without doubt one of the most typical neurodegenerative illnesses. It’s usually thought-about that the prevalence of PD ranges from 100 to 200 per 100,000 individuals and impacts 1% of the inhabitants over the age of 60 years (Tysnes and Storstein, 2017). The lack of dopaminergic neurons within the substantia nigra compact is taken into account the neuropathological attribute of PD. PD is characterised by motor signs together with tremors, rigidity, postural instability, bradykinesia, and non-motor traits resembling autonomic nervous dysfunction, sleep issues, and cognitive and psychological issues (Chen et al., 2020).

As we all know, PD is recognized primarily based mostly on medical options. Subsequently, this can be very troublesome to make a exact analysis in its early stage. Though research have targeted on the identification of potential biomarkers for PD, resembling neuroimaging, cerebrospinal fluid, serum, and saliva biomarkers, there’s nonetheless an absence of dependable biomarkers for the early analysis of PD (Ba and Martin, 2015; Marques et al., 2017; Thobois et al., 2019). Subsequently, it’s of nice curiosity to determine newly non-invasive biomarkers and therapeutic targets for PD.

Round RNAs (circRNAs) are lately found non-coding RNAs (ncRNAs) which can be generated by means of backsplicing, during which the 5′ and three′ ends of the RNA molecule are covalently related (Kristensen et al., 2019). They’ve excessive stability and resistance to degradation as a consequence of their round form and absence of a free finish (Suzuki et al., 2006). It has been demonstrated that circRNAs exhibit distinct properties resembling tissue specificity and stability in each intracellular and extracellular contexts, that are readily accessible and quantifiable in bodily fluids resembling cerebrospinal fluid, blood, plasma, and saliva (Wang et al., 2021). These traits of circRNAs counsel that they could be used as novel non-invasive biomarkers. The truth is, a number of research have recognized a couple of circRNAs in neurodegenerative illnesses resembling Alzheimer’s illness (Akhter, 2018) and a number of system atrophy (Mehta et al., 2020). It’s price mentioning that there are presently few experiences on circRNAs in PD. Though some circRNAs have been recognized that could be dysregulated in PD (Zhong et al., 2021), the networks and underlying mechanisms concerned in circRNA-mediated regulation of PD nonetheless should be explored.

On this research, first, the worldwide expression ranges of the circRNAs in sufferers with PD and wholesome controls have been in contrast by means of microarray, then the outcomes have been validated in one other unbiased PD cohort. Thereafter, candidate circRNAs have been additional analyzed. The potential miRNAs sponged by candidate circRNAs and their goal genes have been analyzed by bioinformatic evaluation. Moreover, the circRNA–miRNA–mRNA regulatory networks, protein–protein interplay, and transcription issue–circRNA interplay have been constructed for predicting the organic operate of the circRNAs for PD.

Supplies and Strategies

Sufferers and Samples

We recruited 63 sufferers With PD and 60 wholesome people From the First Affiliated Hospital of Guangxi Medical College Between January 2019 and June 2021. Sufferers With PD Had been recognized independently by Two neurologists based mostly on the UK Parkinson’s Illness Mind Financial institution Standards (Hughes et al., 1992). the management teams consisted of wholesome individuals matched With PD by way of gender and age and Had no recognized neurological illness, comorbidities, or PD household historical past. the Ethics Committee of the First Affiliated Hospital of Guangxi Medical College accredited this research. All members offered knowledgeable consent. the medical and demographic traits of all members Are outlined in Desk 1. A complete of 123 peripheral blood samples Had been obtained From sufferers With PD and wholesome people and saved vertically at −80°C till complete RNA isolation.


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Desk 1. The highest 5 upregulated and downregulated otherwise expressed circRNAs in PD group and management group.

RNA Isolation and Actual-Time Quantitative Polymerase Chain ReactionTRIzol reagent package (Invitrogen, Takara, Japan) Was used to extract complete RNA From blood samples in response to the protocol offered by the producer. After extraction, complete RNA Was reverse transcribed Into cDNA (RR047A, Takara, Japan). the RT-qPCR Was carried out on the ABI PRISM 7500 (RR820A, Takara, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Was used as an inner reference to normalize the qPCR knowledge. the two–ΔΔCT technique Was used to calculate the relative expression of circRNAs. the primers Are described in Supplementary Desk 1.

CircRNA Microarray and Evaluation

Pattern preparation and microarray hybridization have been carried out in response to the protocol of the Arraystar. KangChen Bio-tech Inc. (Shanghai, China) carried out the Arraystar Human circRNA Array model 2 evaluation. To summarize, circRNAs from every pattern have been enriched by eradicating linear RNAs after which amplified following the producer’s directions. The labeled circRNAs have been hybridized onto the Arraystar and scanned by the Agilent Scanner. CircRNAs exhibiting a fold change (FC) ≥1.5 with a p-value ≤ 0.05 have been thought-about considerably completely different. Volcano plot filtering and fold change filtering have been used to determine differentially expressed circRNAs. The distinct circRNA expression patterns amongst samples have been analyzed utilizing hierarchical clustering. The circRNA microarray uncooked knowledge have been saved within the Gene Expression Omnibus (GEO) (sequence quantity: GSE198273).

Standards for Deciding on circRNA for Additional Bioinformatics Evaluation

The world beneath the curve (AUC) of the receiver working traits (ROC) is used to evaluate the accuracy of biomarkers. The AUC is close to 1, which implies a super mannequin (Pepe et al., 2009). The next pointers are sometimes used to estimate the specificity and sensitivity of a biomarker, e.g., wonderful scored 0.9–1.0; good scored 0.8–0.9; acceptable scored 0.7–0.8; whereas poor scored 0.6–0.7 and fail scored 0.5–0.6 (Xia et al., 2013; Ren et al., 2018). The circRNA stage with an AUC > 0.70 was chosen to proceed ahead with bioinformatics evaluation to analyze potential features.

CircRNA–miRNA–mRNA Interplay Networks

We constructed circRNA–miRNA–mRNA networks in Cytoscape based mostly on circRNA microarray knowledge and the RT-qPCR outcomes. The circRNA–miRNA interplay was predicted utilizing Arraystar’s miRNA goal prediction software program. The Cytoscape plug-in ClueGO (2.5.8) and CluePedia (1.5.8) have been used to foretell goal genes of the highest 5 putative miRNAs for the candidate circRNAs, and the highest 200 goal genes have been screened for the development of the circRNA–miRNA–mRNA networks.

Purposeful Enrichment Analyses

Gene Ontology (GO) enrichment evaluation was used to ascertain useful annotations of miRNA goal genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment evaluation was used to discover the features of those differentially expressed genes. Statistical analyses and visualizations have been performed in R 3.6.3 through the use of the clusterProfiler bundle and the org.Hs.eg.db bundle.

Protein–Protein Interplay Community Evaluation

The protein–protein interplay (PPI) community was created through the use of the Retrieval of Interacting Genes (STRING) on-line instrument to guage the interactions among the many goal genes that have been recognized within the circRNA-miRNA-mRNA networks. The hub genes have been screened from the PPI community utilizing the cytoHubba plugin.

Transcription Issue Prediction

To look at the upstream regulation mechanism of the candidate circRNAs, transcription elements for circRNAs have been screened utilizing the TRCirc database.

Statistical Analyses

Statistical analyses have been performed utilizing SPSS 20.0 software program (IBM, Armonk, NY, United States). The continual variables have been introduced as imply ± normal deviation (SD). The variations in age and intercourse between sufferers with PD and wholesome controls have been assessed utilizing the chi-square (χ2) take a look at or Pupil’s t-test. ROC curves have been used to evaluate the potential biomarkers of the candidate circRNAs. Two-tailed p-values < 0.05 have been thought-about considerably distinction.

Outcomes

A Abstract of the circRNA Microarray Knowledge Profile

Human circRNA microarray was used to display screen dysregulated circRNAs from three sufferers with PD and three wholesome controls. The imply age was 62.67 ± 8.36 years for the PD group and 63.00 ± 8.67 years for controls. Each teams have two male members and one feminine participant. The expression options of the dysregulated circRNAs have been depicted in a two-dimensional hierarchical clustering heatmap (Determine 1A). A volcano plot (Determine 1B) and a scatter diagram (Determine 1C) have been additionally utilized to indicate the variations in circRNA expression between each teams. In keeping with microarray knowledge analyses, a complete of 10,145 circRNAs have been recognized, with 189 antisense (1.86%), 8,518 exonic (83.96%), 73 intergenic (0.72%), 700 intronic (6.90%), and 665 sense overlapping (6.55%) (Determine 1D). After scanning and normalization, 139 differentially expressed circRNAs have been recognized with a threshold of fold-change (FC) absolute worth ≥1.5 and P < 0.05. Of them, 78 circRNAs have been upregulated and 61 have been considerably downregulated. The highest 5 upregulated and downregulated circRNAs are introduced in Desk 2.


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Determine 1. Overview of the round RNA (circRNA) microarray expression knowledge. (A) Hierarchical clustering of the differentially expressed circRNA expression knowledge within the PD group and management group, and upregulated and downregulated circRNAs are coloured in pink and inexperienced, respectively. (B) Volcano plot of the differentially expressed circRNAs within the PD group and management group. The vertical inexperienced traces point out a 2.0-fold (log2-scaled) up and down, respectively. The horizontal inexperienced line represents P = 0.05 (–log10-scaled). The pink squares within the plot signify the differentially expressed circRNAs with statistical significance. (C) The scatter plot reveals the variation in circRNA expression between the PD group and management group. The X and Y values on the scatter plot are the common normalized sign values, proven on a log2 scale. The inexperienced fold-change traces signify 2-fold modifications, thus circRNAs above or under the inexperienced traces point out >2.0-fold upregulation or downregulation. (D) Classification of circRNAs within the PD group and management group.


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Desk 2. Demographic and medical traits of PD group and wholesome controls.

CircRNAs Are Differentially Expressed within the Peripheral Blood of Sufferers With Parkinson’s Illness and Have the Potential as Biomarkers for Parkinson’s Illness

A complete of 10 candidate circRNAs (5 upregulated and 5 downregulated) have been retrieved for additional verification in an unbiased cohort of 63 sufferers with PD and 60 wholesome controls. The medical traits of this cohort are described in Desk 1. The imply age was 60.16 ± 10.51 years for the PD group and 59.25 ± 10.20 years for controls. Statistical analyses confirmed no vital distinction in intercourse or age between each teams (χ2 = 0.004, p = 0.951; t = 0.486, p = 0.628, respectively). The typical UPDRS III rating was 31.30 ± 12.43 for the PD group, and the illness period was 4.44 ± 3.61 years. Enrolled sufferers had a median age of onset of 60.16 ± 10.51 years, and the imply medical Hoehn and Yahr stage was 2.37 ± 0.77.

The ten candidate circRNAs have been verified utilizing divergent primers reasonably than the usual convergent primers by way of RT-qPCR. Of the ten circRNAs, 4 have been confirmed as differentially expressed between each teams (Figures 2A–J). The circRNAs hsa_circRNA_103730, hsa_circRNA_101275, and hsa_circRNA_038416 (Figures 2A–C) have been considerably upregulated, whereas hsa_circRNA_102850 (Determine 2D) was downregulated in sufferers with PD when in comparison with wholesome controls. Contemplating that the incidence of male sufferers with PD is larger than feminine sufferers, we carried out a stratification evaluation based mostly on intercourse. Nonetheless, the outcomes didn’t present a big distinction between female and male sufferers with PD (Supplementary Determine 1A–J).


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Determine 2. Expression profile and ROC curves for circRNAs in sufferers with PD and wholesome controls peripheral blood pattern. (A–J) The relative expression stage of ten candidate circRNAs in sufferers with PD and wholesome management utilizing RT-qPCR detection. (Ok–O) ROC evaluation of the 4 differentially expressed circRNAs in peripheral blood samples. PD, Parkinson’s illness; management, wholesome controls; circRNA, round RNA; hsa, homo sapiens; AUC, the world beneath the curve; TPR, true constructive charge; FPR, false-positive charge. ***p-value < 0.001.

The diagnostic worth of the differentially expressed circRNAs in PD was carried out utilizing the ROC curve. The AUC for hsa_circRNA_103730, hsa_circRNA_102850, hsa_circRNA_101275, and hsa_circRNA_038416 for PD was 0.701, 0.801, 0.843, and 0.674, respectively (2K–N). A circRNA panel combining the 4 candidate circRNAs confirmed the next diagnostic skill to differentiate sufferers with PD from wholesome controls (AUC = 0.938) (Determine 2O).

CircRNA–miRNA–mRNA Interplay Community Development and Transcription Issue–circRNA Interplay Prediction

Usually, ncRNAs act as competing endogenous RNAs (ceRNAs) that regulate goal RNA transcripts by binding competitively to shared miRNAs. It has been nicely established that circRNAs have many miRNA-binding websites which will act as a ceRNA sponge for miRNAs. Contemplating the circRNA with an AUC > 0.70 was thought-about with enough specificity and sensitivity, due to this fact, hsa_circRNA_102850, hsa_circRNA_101275, and hsa_circRNA_103730 have been chosen for the circRNA-regulatory community prediction. Interactions between the three candidate circRNAs and their relationships with miRNAs and goal genes have been predicted by constructing a unified interaction-network mannequin. The interactions between circRNAs and miRNAs have been predicted by the miRNA goal prediction software program. The highest 5 putative miRNAs of the three candidate circRNAs have been chosen to foretell goal genes, and the highest 200 goal genes have been used to assemble the circRNA–miRNA–mRNA interplay networks (Determine 3). The outcomes indicated that the candidate circRNAs could act as endogenous RNAs for modulating goal gene expression.


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Determine 3. The visualization of the circRNA–miRNA–mRNA regulation community. The circRNA–miRNA–mRNA community was constructed based mostly on three circRNAs, 15 miRNAs, and 200 mRNAs. circRNAs have been plotted as purple ellipses, miRNAs have been plotted as orange hexagons, and mRNAs have been plotted as blue squares. miR, microRNA; circRNA, round RNA.

The TRCirc was used to ascertain a transcription issue–circRNA (TF–circRNA) community to discover the exact molecular mechanism of transcriptional regulation of the candidate circRNAs. Hsa_circRNA_103730 (circBase ID: hsa_circ_0005654) and hsa_circRNA_101275 (circBase ID: hsa_circ_0030428) have been predicted to be regulated by concentrating on TH-relevant transcription elements resembling GATA2 and GATA3 (Polanski et al., 2010).

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Enrichment Evaluation of the Differentially Expressed circRNAs

The GO and KEGG enrichment analyses have been performed to analyze the organic operate of the candidate circRNAs. The GO evaluation indicated that the 200 mRNAs have been primarily enriched in detrimental regulation of phosphorylation, mobile response to environmental stimulus, response to a steroid hormone, transcription issue complicated, collagen-containing extracellular matrix, and nuclear transcription issue complicated (Determine 4A). The PI3K–Akt signaling pathway and MAPK signaling pathway have been discovered to have vital enrichment within the KEGG pathway evaluation (Determine 4B).


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Determine 4. The GO and KEGG pathway enrichment evaluation for 200 mRNAs. (A) The foremost enriched and significant GO phrases. (B) The highest 20 enrichment scores within the KEGG pathway evaluation. GO, Gene Ontology; BP, organic processes; CC, mobile parts; MF, molecular features; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Protein–Protein Interplay Community Development

We constructed a PPI community in response to circRNA–miRNA–mRNA interplay evaluation. The PPI networks recognized 107 proteins and 200 edges of the candidate circRNA networks that could be concerned in PD (Determine 5A). We used the cytoHubba plugin to search out the crucial genes within the means of PD pathogenesis and obtained a sub-network containing 10 genes, together with ESR1, PTEN, SHC1, IGF1R, SMAD2, KRAS, MDM2, HIF1A, BMP4, and ACVR2B (Determine 5B). The outcomes of PPI interplay networks confirmed that ESR1, PTEN, and SHC1 could also be concerned in PD as a very powerful targets.


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Determine 5. The PPI community for 200 mRNAs. (A) A PPI community composed of 107 proteins and 200 edges. (B) The sub-network containing 10 hub genes. Proteins are plotted as circles and given completely different colours, the darker the colour, the extra vital the protein is within the interplay community. PPI, protein–protein interplay.

Dialogue

Parkinson’s illness (PD) is an irreversible neurodegenerative illness (Hayes, 2019). The exact pathogenic mechanisms underlying PD stay largely unknown. Many research have been undertaken in recent times to supply new insights into PD however primarily concentrate on protein-coding genes or miRNAs (Goh et al., 2019; Rathore et al., 2021). CircRNAs have attracted a lot consideration for his or her particular traits resembling conserved, tissue- or cell-type particular, and involvement with miRNA regulation in a number of issues resembling cardiovascular illnesses (Altesha et al., 2019), most cancers (Chen and Shan, 2021), and diabetes (Sakshi et al., 2021), however their roles in PD stay unclear.

On this research, we first used microarray analyses to acquire the expression profiles of circRNAs in sufferers with PD and wholesome controls. A complete of 139 dysregulated circRNAs have been recognized. Differential expression evaluation in an unbiased PD cohort revealed that hsa_circ_101275, hsa_circ_038416, hsa_circ_103730, and hsa_circ_12850 is perhaps used as potential novel biomarkers for PD. In addition to, a circRNA panel combining the above 4 differentially expressed circRNA confirmed the next diagnostic skill to differentiate sufferers with PD from controls (AUC = 0.938). Though a earlier research has recognized that some circRNAs have been differentially expressed in sufferers with PD and controls (Zhong et al., 2021), the networks and underlying mechanisms concerned in circRNA-mediated regulation of PD haven’t been explored.

To research the potential operate of the differentially expressed circRNAs in PD, we constructed a core circRNA–miRNA–mRNA regulatory community, protein–protein interplay, and transcription issue–circRNA interplay analyses. The GO and KEGG analyses confirmed that these candidate circRNAs have been enriched in PI3K–Akt signaling and MAPK signaling pathways. The disruption of Akt-mediated sign transduction contributes to the pathogenesis of a number of neurodegenerative illnesses (Rai et al., 2019), together with PD (Cao et al., 2017; Chen et al., 2017; Hu et al., 2018). As an illustration, a current research has indicated that amentoflavone protects dopaminergic neurons in opposition to MPTP/MPP+-induced neurotoxicity, presumably by activating the PI3K/Akt signaling pathways (Cao et al., 2017). Whether or not these candidate circRNAs are concerned in PD by means of the PI3K–Akt signaling pathway must be additional investigated.

Usually, circRNAs function miRNA sponges, adsorbing miRNAs and altering miRNA expression ranges, therefore influencing downstream goal gene expression (Moreno-García et al., 2020). Subsequently, we constructed circRNA–miRNA–mRNA regulatory networks and recognized a number of miRNAs resembling miR-29b-2-5p, miR-22-3P, miR-141-3p, and miR-18b-5p within the candidate circRNA–miRNA–mRNA regulatory networks. What must be famous is that one of the vital PD pathogenic gene GBA (Riboldi and Di Fonzo, 2019) and its pseudogene GBAP1 are miR-22-3P goal genes (Straniero et al., 2017). One other research discovered miR-22 was an vital goal for suppressing apoptosis and the era of reactive oxygen species in a mobile mannequin of PD (Yang et al., 2016). Whether or not these candidate circRNAs can act as a sponge for miR-29b-2-5p and miR-22-3P by modulating the GBA axis and the apoptosis cascade must be additional explored.

We established circRNA–miRNA–mRNA regulatory networks and recognized 10 hub genes (ESR1, PTEN, SHC1, IGF1R, SMAD2, KRAS, MDM2, HIF1A, BMP4, and ACVR2B) that have been intently associated to PD through the use of PPI community evaluation. The recognized hub genes may present vital targets within the pathogenesis of PD. For instance, genetic research have discovered ESR1 gene polymorphisms could also be linked to PD susceptibility (Handel et al., 2013), IGF1R was found to have anti-inflammatory properties in a rat mannequin of PD (Xu et al., 2009; Du et al., 2021), BMP4 can induce the phenotypic of striatal dopaminergic neurons in vitro (Stull et al., 2001). General, the findings additional assist the significance of the recognized circRNAs in PD, although the precise mechanisms are nonetheless unclear.

At current, circRNA research are primarily targeted on its downstream mechanisms, however upstream mechanisms, resembling how circRNAs are generated, are nonetheless missing. Transcription issue Twist1 has been recognized to transcriptionally regulate the expression of circRNA-10720 (Meng et al., 2018), and one other transcription issue c-Myc might speed up the circularization and biogenesis of circUHRF1 (Zhao et al., 2020), suggesting that transcription elements could also be concerned within the circRNA upstream regulation course of. We used TRCirc to ascertain a transcription elements–circRNA (TF-circRNA) community and located hsa_circRNA_103730 and hsa_circRNA_101275 have been predicted to be regulated by TH-relevant transcription elements resembling GATA2 and GATA3 (Polanski et al., 2010), which additional expanded the operate of the 2 PD-related transcription elements.

In abstract, our outcomes counsel the circRNA panel (hsa_circ_101275, hsa_circ_038416, hsa_circ_103730, and hsa_circ_12850) and the established circRNA–miRNA–mRNA regulation networks may present potential novel biomarkers and therapeutic targets for PD.

Knowledge Availability Assertion

The datasets introduced on this research could be present in on-line repositories. The names of the repository/repositories and accession quantity(s) could be discovered within the article/Supplementary Materials.

Ethics Assertion

The research involving human members have been reviewed and accredited by the Ethics Committee of The First Affiliated Hospital of Guangxi Medical College. The sufferers/members offered their written knowledgeable consent to take part on this research.

Creator Contributions

YX and JJL wrote the article and labored with HC on the conception and design. QZ and HH carried out the experiments. JL and JH analyzed the info. QO and JW polished the article. All authors contributed to the article and accredited the submitted model.

Funding

This research was supported by the Nationwide Pure Science Basis of China (Nos. 82060248 and 81801268), Guangxi Medical and Well being Acceptable Expertise Improvement and Utility Venture (No. S2019095), Scientific Analysis “Climbing” Program of The First Affiliated Hospital of Guangxi Medical College (No. YYZS2020017), Innovation Venture of Guangxi Graduate Training (No. YCSW2021117), and “Medical Excellence Award” funded by the Inventive Analysis Improvement Grant from The First Affiliated Hospital of Guangxi Medical College.

Battle of Curiosity

The authors declare that the analysis was performed within the absence of any business or monetary relationships that may very well be construed as a possible battle of curiosity.

Writer’s Notice

All claims expressed on this article are solely these of the authors and don’t essentially signify these of their affiliated organizations, or these of the writer, the editors and the reviewers. Any product that could be evaluated on this article, or declare that could be made by its producer, will not be assured or endorsed by the writer.

Acknowledgments

Microarray experiments have been carried out by KangChen Bio-tech Inc., Shanghai, China.

Supplementary Materials

The Supplementary Materials for this text could be discovered on-line at: https://www.frontiersin.org/articles/10.3389/fnins.2022.893713/full#supplementary-material

Supplementary Determine 1 | Relative expression of candidate circRNAs in male sufferers with PD and feminine sufferers with PD. PD, Parkinson’s illness; management, wholesome controls; circRNA, round RNA; hsa, homo sapiens. The stratification analyses of the ten candidate circRNAs in sufferers with PD and wholesome management in response to intercourse (Supplementary Determine 1A–J).

Footnotes


References

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