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Frontiers | Plasma miR-153 and miR-223 Ranges as Potential Biomarkers in Parkinson’s Illness


Introduction

Parkinson’s illness (PD) is a typical neurodegenerative illness that often impacts the aged (Ascherio and Schwarzschild, 2016; Poewe et al., 2017) and brings a heavy financial burden to the sufferers’ households and society. Pathologically, it’s characterised by the selective lack of dopaminergic neurons within the substantia nigra and Lewy our bodies within the remaining neurons (Homayoun, 2018). At present, the medical prognosis of PD is especially primarily based on medical examination and neuroimaging analysis (Postuma et al., 2015b). Misdiagnosis and missed prognosis typically happen in medical observe. Due to this fact, the identification of early goal biomarkers is among the essential priorities in PD analysis.

As one sort of endogenous non-coding RNA molecules, small molecule RNAs (miRNAs) can inhibit protein-coding genes by affecting mRNA translation and circulation within the blood and cerebrospinal fluid (CSF) and are concerned within the pathogenesis of many ailments, together with PD (Ramaswamy et al., 2018; Goh et al., 2019; Van den Berg et al., 2020). The best biofluid pattern is CSF due to its shut interplay with the pathological mind. Nevertheless, CSF can solely be obtained via a lumbar puncture, which can’t be nicely accepted by the sufferers (Starhof et al., 2019). Blood is comparatively straightforward to entry. Blood cells may partly replicate the physiological and pathological stimuli and coverings due to their particular mobile organizations and connections with many of the tissues within the physique together with the mind (Soreq et al., 2014). Intriguingly, it has been reported that the transcriptomic patterns of mind and peripheral blood overlap by roughly 81.9% (Liew et al., 2006). Due to this fact, evaluation of miRNAs in blood might be one sensible technique to establish biomarkers in PD (Arshad et al., 2017).

The significance of α-synuclein (SNCA) in PD is seminal. Mutations within the SNCA gene had been the primary reported genetic sorts in acquainted PD (Polymeropoulos et al., 1997). Furthermore, SNCA is the most important part of Lewy our bodies in sporadic PD and it has been demonstrated that the expression ranges of SNCA are elevated in sporadic sufferers with PD, in addition to in animal fashions with PD (Spillantini et al., 1998; Vila et al., 2000). Curiously, greater ranges of regular non-mutated SNCA may additionally induce SNCA aggregation and Lewy physique formation in PD, suggesting that whatever the mutational standing of the SNCA gene, even small adjustments in SNCA ranges may set off PD growth and development (Singleton et al., 2003). Due to this fact, the expression ranges of SNCA might play an essential function in triggering PD development and might be potential biomarkers for PD.

A number of miRNAs had been reported to have the ability to regulate SNCA gene expression, and the particular ones are miR-7, miR-153, and miR-223, as predicted by three miRNA goal gene prediction software program packages (TargetScan Human 7.1, MicroCosm Targets). A current examine reported that two SNCA-related miRNAs, miR-153 and miR-223 ranges, had been decreased within the saliva of sufferers with PD, and will function promising PD biomarkers (Cressatti et al., 2020). Whereas their corresponding expression ranges within the blood of sufferers with PD had been elusive. On this examine, we detected the expression ranges of three SNCA associated miRNAs (miR-7, miR-153, and miR-223) in plasma and explored their diagnostic worth and associations with the medical phenotype in PD.

Supplies and Strategies

Topics

This examine was authorised by the Medical Ethics Committee of Shanghai Ninth Individuals’s Hospital, Shanghai Jiao Tong College College of Medication, Shanghai, China. All of the members supplied written knowledgeable consent. From September 2019 to September 2021, seventy-five sufferers with PD and 73 regular controls (NCs) had been included on this examine. Sufferers with PD had been examined and recognized by at the very least two skilled neurologists from the Division of Neurology of our hospital, based on the Motion Dysfunction Society (MDS) medical diagnostic standards of PD (Postuma et al., 2015a,b). NCs had been recruited from the well being examination heart of our hospital, they usually had no apparent neurological issues equivalent to stroke, mind tumor, epilepsy, parkinsonism, associated issues, and so forth. Each the members within the PD and NC teams had been matched for age, gender, and Chinese language model of the Mini-mental State Examination (MMSE) rating.

Whole miRNA Extraction From Plasma

In complete, two milliliters of peripheral blood had been drawn from the cubital vein right into a vacuum blood tube with ethylenediaminetetraacetic acid (EDTA) after which the plasma was aliquoted (200 μl) right into a sterile tube and saved at −80°C. Plasma samples of members had been collected to extract plasma miRNAs. Whole miRNA was extracted and purified from plasma samples utilizing the miRNeasy Serum/Plasma Equipment (Qiagen, Hilden, Germany, Catalog quantity 217184). All of the operations had been carried out in strict accordance with the producer’s directions. The artificial Caenorhabditis elegans miR-39 (cel-miR-39, Qiagen; 219610) was added to plasma after lysis as an exterior reference, which is devoid of sequence homology to human miRNAs.

Reverse Transcription and Actual-Time Quantitative Polymerase Chain Response Experiment

RNA was reverse transcribed with a miScript reverse transcription equipment (Qiagen, Catalog quantity 218161). The expression of miRNAs was calculated by real-time quantitative polymerase chain response (RT-qPCR) utilizing the miScript SYBR Inexperienced PCR equipment (Qiagen, Catalog quantity 218073). The American ABI7500 Fluorescent Quantitative PCR instrument was used for detection. Particular miRNA primers had been obtained from TIANGEN Biotech Co., Ltd. (Beijing, China). The calculation of the relative miRNA expression ranges was analyzed utilizing the delta–delta cycle threshold worth (2–ΔΔCt) methodology. The expression ranges of various miRNAs had been normalized with cel-miR-39 and in contrast as described beforehand (Schmittgen and Livak, 2008). The expression ranges of miR-7, miR-153, and miR-223 in sufferers with PD and NCs had been in contrast.

Scientific Analysis

For the sufferers with PD, motor severity was measured with a modified Hoehn and Yahr (H&Y) stage (Hoehn and Yahr, 1967) and Unified Parkinson’s Illness Ranking Scale half III (UPDRS-III) (Richards et al., 1994). medical attainable speedy eye motion sleep conduct dysfunction (cpRBD) was screened through the use of REM Conduct Dysfunction Screening Questionnaire (RBDSQ) (Nomura et al., 2011). Freezing of gait (FOG) was thought-about current when topics had multiple rating on merchandise 3 of the Freezing of Gait questionnaire (FOG-Q) (Giladi et al., 2000). We used MMSE (Katzman et al., 1988) and the Chinese language Model of the Montreal Cognitive Evaluation-Fundamental (MoCA-BC) (Xu et al., 2021) to measure sufferers’ cognitive operate. Autonomic dysfunction was assessed by the Scales for Outcomes in PD Autonomic Dysfunction (SCOPA-AUT) (Verbaan et al., 2007). Olfactory operate was assessed by SS-16 as within the earlier report (Chen et al., 2012). The severity of depressive signs was assessed by the 17-item Hamilton Ranking Scale for Despair (HAMD-17), and a rating of ≥8 was thought to be melancholy (Hamilton, 1960). Newly recognized, drug naïve sufferers had been thought to be de novo PD. For medicated sufferers, levodopa equal day by day dose (LEDD) was calculated.

Statistical Evaluation

SPSS model 23.0 (IBM Company, Armonk, NY, United States) was used for statistical evaluation. Steady variables are expressed because the means ± SD or medians [interquartile ranges (IQRs), Q1–Q3]; categorical variables are expressed as frequencies and percentages. Comparisons of means between the 2 teams had been carried out utilizing the impartial t-test or the Mann–Whitney U check as acceptable. For the reason that distribution of miRNA expression ranges in every group was extremely skewed, the expression degree was log-transformed for regular distribution. The chi-square or Fisher actual check was used for evaluating proportions. The diagnostic worth of every miRNA was evaluated by delineating the receiver working attribute (ROC) curve and deciding on the purpose with the utmost Yuden index because the cut-off worth and the realm beneath the curve (AUC) was calculated accordingly. As well as, the variations between the three miRNAs in PD subtypes (de novo PD vs. medicated PD, cpRBD constructive vs. cpRBD unfavorable, and so forth.) had been additionally in contrast. We analyzed the continual variables by one-way ANOVA or non-parametric Kruskal–Wallis checks, relying upon whether or not the information had been usually distributed or not. The p-values for these three group comparisons had been adjusted utilizing the Bonferroni methodology. Correlations between the three miRNA ranges and medical knowledge (illness period, H&Y stage, UPDRS-III, RBDSQ, and so forth.) in PD had been assessed utilizing the Pearson’s correlation coefficient. A number of linear regression evaluation was used to discover the independently related components of mRNA ranges in PD. The check degree (α) was 0.05.

Outcomes

Normal Traits

The overall traits of sufferers with PD and NCs had been proven in Desk 1. Age, gender, and MMSE rating had been constant between sufferers with PD and NCs. For sufferers with PD, the median illness period was 2.0 years, and 34.7% (n = 26) of the sufferers had been de novo sufferers with PD. In complete, eighteen sufferers (24.0%) had a household historical past of PD or tremors. In complete, forty-six sufferers (61.3%) had H&Y stage ≥2. The frequency of hyposmia and cpRBD had been 66.7 and 49.3%, respectively. 9 sufferers (12.0%) had wearing-off and two sufferers (2.7%) had dyskinesia. Not one of the sufferers obtained deep mind stimulation.


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Desk 1. Normal traits in sufferers with PD and NCs.

Comparability of Relative Expression of miR-7, miR-153, and miR-223 in Plasma Between Sufferers With Parkinson’s Illness and Regular Controls

As proven in Determine 1, the relative expression ranges of miR-153 and miR-223 within the plasma of the sufferers with PD had been considerably decrease than that of the NCs each within the uncooked and the log-transformed worth. Imply miR-153 concentrations for the sufferers with PD was 0.68 relative to NCs (z = −2.757, p = 0.006; Determine 1B). The log-transformed expression ranges of miR-153 was considerably completely different in sufferers’ with PD with plasma when put next with NCs (p = 0.006; Determine 1E). Imply miR-223 concentrations for the sufferers with PD had been 0.18 relative to NCs (z = −7.630, p = 0.000; Determine 1C). The log-transformed expression ranges of miR-223 had been considerably completely different in PD sufferers’ plasma when put next with NCs (p = 0.000; Determine 1F). Whereas the distinction in miR-7 focus and log-transformed expression degree between sufferers with PD and NCs was not statistically vital (0.89 vs. 1.00, z = −0.604, p = 0.546; Figures 1A,D). MiR-153 distinguished sufferers with PD from NCs with 54.8% sensitivity and 74.7% specificity (AUC = 0.631, cut-off worth 0.85; Determine 1G), whereas, miR-223 distinguished sufferers with PD from NCs with 68.5% sensitivity and 88.0% specificity (AUC = 0.862, cut-off worth 0.63; Determine 1H).


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Determine 1. MiR-7, MiR-153, and miR-223 ranges within the plasma of sufferers with PD relative to NCs. Imply expression ranges of miR-7 (A), miR-153 (B), and miR-223 (C) had been decided by real-time quantitative polymerase chain response and analyzed utilizing the delta-delta cycle threshold worth (2– Δ Δ Ct) methodology. Information had been normalized by way of log transformation and reported for miR-7 (D), miR-153 (E), and miR-223 (F). Midline in field and whisker plots depicts the median, with higher and decrease limits representing most and minimal values, respectively. ROC curves had been estimated for miR-153 (G) and miR-223 (H), with AUC indicated. n = 73 and 75 for NCs and PD teams, respectively. AUC, space beneath the curve; NCs, regular controls; miR, microRNA; PD, Parkinson’s illness; ROC, receiver working attribute.

Relationship Between miR-153, miR-223, and Scientific Phenotype in Parkinson’s Illness

Each miR-153 and miR-223 expression ranges weren’t associated to gender or age. Log-transformed miR-153 expression degree in de novo PD was decrease than that within the handled sufferers and in NCs (p = 0.008; p < 0.001; respectively; Determine 2A). Imply miR-153 concentrations for de novo PD are 0.51 and for handled PD is 0.68 relative to NCs. MiR-153 expression degree elevated regularly with illness period (r = 0.358, p = 0.002; Determine 2B) in PD.


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Determine 2. Relationship between miR-153, miR-223, and medical phenotype in PD. Expression ranges of miR-153 in NCs handled PD and de novo PD teams (A). Correlation evaluation between miR-153 and illness period (B). Correlation evaluation between miR-153 and UPDRS-III (C). Correlation evaluation between miR-153 and H&Y stage (D). Expression ranges of miR-223 in NCs, PD with cpRBD, and PD with out cpRBD teams (E). Correlation evaluation between miR-223 and RBDSQ (F). NCs, regular controls; miR, microRNA; PD, Parkinson’s illness; UPDRS-III, Unified Parkinson’s Illness Ranking Scale half III; H&Y stage, Hoehn and Yahr stage; cpRBD, medical attainable speedy eye motion sleep conduct dysfunction; RBDSQ, speedy eye motion sleep conduct dysfunction screening questionnaire.

Regarding motor signs, we discovered that the miR-153 expression degree was positively related to UPDRS-III scores (r = 0.264, p = 0.022; Determine 2C) and was marginal correlated to the H&Y stage (r = 0.209, p = 0.072; Determine 2D).

Relating to non-motor signs, log-transformed plasma miR-223 expression degree was decreased in sufferers with cpRBD in contrast with these with out cpRBD and NCs (p = 0.021; p < 0.001; respectively; Determine 2E), Imply miR-223 concentrations for sufferers with cpRBD is 0.08 and for these with out cpRBD is 0.27 relative to NCs and its degree was negatively related to RBDSQ rating (r = -0.334, p = 0.003; Determine 2F). The imply MOCA-BC rating for the sufferers with PD with cpRBD sufferers was decrease than these with out cpRBD (21.00 vs. 24.00, p = 0.004).

A number of linear regression evaluation revealed that illness period (p = 0.049) was the one impartial related issue of miR-153 degree; whereas, solely RBDSQ (p = 0.009) was associated to miR-223 degree in sufferers with PD.

Dialogue

As a typical molecular biomarker for illness prognosis, plasma miRNAs obtain nice consideration within the analysis of PD. That is the primary examine that comprehensively analyzed the expression ranges of three SNCA-related miRNAs: miR-7, miR-153, and miR-223 in plasma and explored their diagnostic worth in sufferers with PD by way of serial scales. We discovered that (1) lowered expression ranges of miR-153 and miR-223 in PD relative to wholesome controls; (2) miR-153 degree correlated with illness period; whereas, the miR-223 degree was associated to RBD in sufferers with PD.

Outcomes of this examine confirmed that the plasma ranges of miR-153 and miR-223 had been decreased in PD, they usually distinguished PD from NC with 63.1 and 86.2% accuracy, respectively, indicating these two miRNAs as potential diagnostic biomarkers of idiopathic PD. Earlier research on miR-153 and miR-223 expression ranges in PD vs. NC in several biofluids are summarized in Desk 2; Vallelunga et al. (2014),Gui et al. (2015), Zhang et al. (2017), Mancuso et al. (2019), Cressatti et al. (2020). Totally different specimen sources and pattern sizes might contribute to the inconsistency of the outcomes. Our discovering is in keeping with the outcomes from human saliva. Cressatti et al. (2020) additionally discovered miR-153 and miR-223 ranges had been considerably decreased within the saliva of human sufferers with PD as compared with non-neurological controls. It’s well-known that miR-153 and miR-223 may induce downregulation of SNCA expression by binding the three’ UTR of SNCA. The lowered expression degree of miR-153 and miR-223 in sufferers’ plasma or saliva not directly means that there could also be irregular expression ranges of SNCA in sufferers with PD.


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Desk 2. Serial research on miR-153 and miR-223 expression ranges in PD vs. NC.

A novel discovering is that plasma miR-153 degree in de novo PD was decrease than that in handled sufferers and its degree elevated regularly with illness period and was positively related to UPDRS-III scores. Additional multivariate evaluation confirmed that illness period is the one impartial related issue of miR-153 degree in PD, suggesting that plasma miR-153 degree might characterize illness development in PD. Relating to the analysis associated to miR-153 and illness development, Cressatti et al. (2020) confirmed that the L-dopa dose elevated as a operate of the H&Y stage in sufferers with PD. Nevertheless, log-transformed miR-153 expression ranges weren’t correlated to LEDD (Cressatti et al., 2020). Narasimhan et al. (2014) reported that miR-153 is considerably upregulated in response to a physiologically related degree of paraquat (PQ) and miR-153 suppresses Nrf2/ARE cascade and its related cryoprotection throughout PQ-induced toxicity in dopaminergic neurons. Due to this fact, it was speculated that miR-153 might serve an anti-oxidative stress impact in PD, which can partly clarify the responsive enhance of its degree because the illness development.

One other fascinating discovering is that plasma miR-223 degree was decreased in sufferers with PD with cpRBD in contrast with these with out cpRBD, and its degree was negatively related to RBDSQ rating, indicating that miR-223 is related to RBD subtype in sufferers with PD. Quite a few current research have prompt that sufferers with PD and RBD have a extra diffuse SNCA neuropathology (Postuma et al., 2015a; Leclair-Visonneau et al., 2017). Leclair-Visonneau et al. (2017) confirmed that enteric phosphorylated SNCA histopathology was extra frequent within the subgroup of PD sufferers with RBD in contrast with these with out RBD. PD sufferers with RBD have a higher frequency of SNCA pathology within the enteric nervous system (Leclair-Visonneau et al., 2017). Postuma et al. (2015a) discovered that PD with RBD had elevated SNCA deposition in all mind areas examined, with 9 of ten areas considerably completely different. RBD signs amongst sufferers with PD might have a higher density and vary of SNCA pathology on post-mortem (Postuma et al., 2015a). Many current research have proven that RBD in PD marks a subtype of the illness characterised by an elevated threat of cognitive dysfunction and dementia (Dauvilliers et al., 2018). In our PD sufferers with RBD, the expression degree of miR-223 and imply MoCA-BC rating was decrease than sufferers with out RBD; these knowledge verify earlier outcomes obtained in sufferers with AD and MCI, displaying a big downregulation of miR-223 within the blood (Mancuso et al., 2019). Furthermore, miR-223 induced downregulation of SNCA expression by binding the three’ untranslated area of SNCA, thus might inflicting extra irregular deposition of SNCA in sufferers with PD with RBD.

It needs to be famous that miR-7 is enriched within the mind and has been discovered to inhibit the expression of SNCA, there have been a couple of research on miR-7 as a biomarker for PD prognosis lately (Zhou et al., 2016; Li et al., 2019; Starhof et al., 2019; Cressatti et al., 2020; Ravanidis et al., 2020). Nevertheless, the outcomes had been inconsistent (Supplementary Desk 1). Totally different sources of tissue and biofluid, pattern measurement, and high quality management might partly clarify the inconsistency. Additionally, the connection between the mind and the peripheral bioliquid is complicated. Above all, we speculate that miR-7 can’t distinguish PD from NC in some sources of biofluid, particularly in plasma and saliva, and is probably not used as a diagnostic biomarker of idiopathic PD.

Some limitations needs to be talked about on this examine. The recruited topics got here from one heart, and the pattern measurement is comparatively restricted; we solely in contrast PD with the NC group and didn’t enroll the illness management group equivalent to sufferers with important tremor or a number of system atrophy. As well as, we didn’t detect the expression ranges of those miRNAs in genetic sufferers with PD. It isn’t identified whether or not these miRNAs differ between genetic and idiopathic PD or not. The prognosis of RBD was primarily based on a subjective questionnaire, not on goal polysomnography (PSG). Blood is less complicated to acquire than CSF and saliva within the medical observe. The essential discovering from this examine is that plasma miR-153 and miR-223 ranges might function helpful, non-invasive, and comparatively cheap diagnostic biomarkers of idiopathic PD. A multi-center cohort examine with constant protocols is warranted sooner or later to establish their correlations with SNCA ranges and the diagnostic worth on illness growth and development. Furthermore, whether or not plasma miR-223 degree is decreased in idiopathic RBD and whether or not its degree may predict phenoconversion from idiopathic RBD to PD deserves additional investigation.

Information Availability Assertion

The uncooked knowledge supporting the conclusions of this text will likely be made obtainable by the authors, with out undue reservation.

Ethics Assertion

The research involving human members had been reviewed and authorised by the Medical Ethics Committee of Shanghai Ninth Individuals’s Hospital, Shanghai Jiao Tong College College of Medication, Shanghai, China (2017-303-T223). The sufferers/members supplied their written knowledgeable consent to take part on this examine.

Writer Contributions

LW, QX, MZ, and WC: conceptualization. LW, QX, MZ, and YC: methodology. LW, QX, CJ, YJ, and YL: validation. LW and QX: formal evaluation, writing – authentic draft preparation, and visualization. LW, QX, MZ, YC, CJ, YJ, YL, QH, LZ, and YD: investigation. LW, QH, LZ, YD, JL, and WC: assets. JL and WC: knowledge curation and undertaking administration. QH, LZ, YD, JL, and WC: writing – overview and enhancing. MZ, YC, and WC: supervision. LW and WC: funding acquisition. All authors agreed to the printed model of the manuscript.

Funding

This analysis was funded by the Nationwide Pure Science Basis of China (81401039 and 81501085), Shanghai Pujiang Program (18PJD023), Shanghai Medical Steering Program (17411964000), Scientific Analysis Program of Shanghai Ninth Individuals’s Hospital Affiliated to Shanghai Jiao Tong College College of Medication (JYLJ202003), Challenge of Biobank from Shanghai Ninth Individuals’s Hospital, Shanghai Jiao Tong College College of Medication (YBKB202120), Shanghai Younger Science and Know-how Abilities Crusing Program (18YF1412600), and Elementary Analysis Program Funding of Shanghai Ninth Individuals’s Hospital Affiliated to Shanghai Jiao Tong College College of Medication (JYZZ155).

Battle of Curiosity

The authors declare that the analysis was carried out within the absence of any industrial or monetary relationships that might be construed as a possible battle of curiosity.

Writer’s Be aware

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

Acknowledgments

We wish to thank all the themes that participated within the examine.

Supplementary Materials

The Supplementary Materials for this text could be discovered on-line at: https://www.frontiersin.org/articles/10.3389/fnins.2022.865139/full#supplementary-material

Footnotes


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