MicroRNA as diagnostic biomarker and therapeutic agent for microfibrillar neuropathy

Abstract

The present application describes miRNAs in the diagnosis and treatment of small fiber neuropathy (SFN).

Description

TECHNICAL FIELD

[0001] Small fiber neuropathy (SFN) is a multifactorial disorder that affects both Aδ and C fibers. Typical clinical manifestations include symmetrical, length-dependent and autonomic sensory symptoms, often accompanied by chronic neuropathic pain, which severely affects patients' daily life 1 .

[0002] Due to its complexity and lack of evidence of pathophysiological mechanisms, current therapeutic strategies are still limited to symptomatic relief, with low efficacy and based on trial and error.

[0003] MicroRNAs (miRNAs) represent potential therapeutic targets in complex diseases due to their pleiotropic features and ability to regulate multiple molecular pathways and mediate cell-to-cell communication.

[0004] To date, preclinical and clinical studies have identified several miRNAs associated with axonal degeneration that affect axonal guidance signals and mediate local post-transcriptional and post-translational changes in the axonal microenvironment 2,3 ; however, the role of each miRNA is strongly dependent on the microenvironment in which it is expressed, especially in multifactorial diseases.

[0005] The disclosure of Huang Chen et al, “Micro RNAs in autoimmune liver diseases: from diagnosis to potential therapeutic targets”, BIOMEDICINE & PHARMACOTHERAPY, ELSEVIER, FR, vol. 130, (2020-08-09) and international patent application WO 2016 / 144265 describes microRNA-26b-5p and microRNA-20a-5p. SUMMARY

[0006] The inventors of the present patent application surprisingly identified that, under the pathophysiology of SFN, specific miRNAs are significantly associated with the degree of degeneration of intraepidermal nerve fibers (IENF). BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 The design of the study leading to the present invention is shown.

[0008] Figure 2The morphological and molecular profile in SFN patients skin biopsies is shown. (A) Model of lower limb skin innervation in healthy subjects (HC) and idiopathic SFN patients. Skin biopsies were immunostained with antibodies against phosphoglycerate phosphatase 9.5 (PGP9.5) and observed with bright field microscopy. SFN patients show a reduction of IENF. (B) Unsupervised heat map showing the differences in miRNA expression profile between SFN and HC patients, expressed in ΔCq values.

[0009] Figure 3 The downregulation of miR-26b-5p and miR-20a-5p miRNAs in the epidermis of SFN patients is shown. A) Boxplot of miR-26b-3p in the discovery cohort and validation cohort showing downregulation in SFN patients with respect to HC. Comparison was made by applying the Wilcoxon-Mann-Whitney test. BH-adjusted p values are shown in the graph; B) and C) Correlation of miR-26b-5p expression with delta IENFD values of subjects enrolled in the discovery step (B) and validation step (C). D) Correlation of miR26b-5p and miR20a-5p expression. E) Boxplot of miR-20a-5p in the discovery cohort and validation cohort showing downregulation in SFN patients with respect to HC. Comparison was made by applying the Wilcoxon rank sum test. Because this miRNA did not pass the multiple testing correction analysis, unadjusted p values are reported, F) and G) Spearman's correlation of miR-20a-5p expression with delta IENFD values of subjects enrolled in the discovery step (F) and validation step (G). Spearman's correlation coefficient and p values are shown in the graph. H) ROC analysis showing significant discriminative power for SFN.

[0010] Figure 4 The Pearson correlation showing the functional relationship between miR-20a-5p and miR-26b-5p miRNAs and their target genes is shown.

[0011] Figure 5 The in situ hybridization showing that miR-26b-5p is mainly expressed in the epidermis is shown.

[0012] OBJECT OF THE INVENTION In a first object, the use of miRNAs in the diagnosis of small fiber neuropathy (SFN) is described.

[0013] In a second object, the medical use of the miRNAs of the present invention in the treatment of small fiber neuropathy (SFN) is described.

[0014] In a third object, the use of the same miRNAs in the identification of compounds for the treatment of small fiber neuropathy is described. DETAILED DESCRIPTION

[0015] According to a first object, the present application describes the use of miRNAs in the diagnosis of small fiber neuropathy (SFN).

[0016] In particular, said miRNAs are:

[0017] In a first aspect of the present application, when one or both of the above two miRNAs are identified and downregulated, a diagnosis of small fiber neuropathy is made.

[0018] In a preferred aspect, when miR-26b-5p is downregulated, a diagnosis of small fiber neuropathy is made.

[0019] In a more preferred aspect, when at least miR-26b-5p (possibly also including miR-20a-5p) is identified and downregulated, a diagnosis of small fiber neuropathy is made.

[0020] In particular, the concentration of said miRNAs is measured in an isolated sample representative of the epidermis of a patient suspected of suffering from said disease.

[0021] More in particular, the fact that they are downregulated is determined with reference to healthy controls (HC).

[0022] In a second object, the medical use of the miRNAs of the present application in the treatment of small fiber neuropathy (SFN) is described.

[0023] To this end, each of the above two miRNAs can be used for the treatment, also using them together.

[0024] In a third object, the use of the same miRNAs in the identification of compounds for the treatment of small fiber neuropathy is described.

[0025] Purpose of the study and design In the present study, an epidermal molecular profile associated with a severe degeneration of the density of intraepidermal nerve fibers (IENF) was identified. Skin biopsy samples were collected from SFN patients and healthy subjects (HC). The miRNA profiling analysis was performed in two independent steps: an exploratory step (N=13) and a validation step (N=20), which allowed the identification of two miRNAs closely related to each other, which were significantly differentially expressed in SFN patients. Gene expression profiling at mRNA level was performed and correlation analysis with miRNA expression levels was performed to investigate the effect of the two candidate miRNAs on their target transcripts.

[0026] In situ hybridization was used to identify the cellular location of miRNAs. Figure 5 Furthermore, the homogeneity selection of this cohort is an important factor in identifying epidermal-specific miRNAs and characterizing small fiber neuropathy, and can serve as an additional biomarker in clinical trials.

[0027] Subject recruitment Patients were recruited consecutively (n=18) from the SFN clinical clinic of the Carlo Besta Foundation for Neurological Research (IRCCS) (Milan, Italy). Healthy controls (HC, n=15) were recruited under the PAIN-Net project (Grant No. 721841).

[0028] The inclusion criteria for idiopathic SFN are: presence of pain or sensory disturbances, with both length-dependent and diffuse distribution; decreased intradermal nerve fiber density; and no underlying clinical symptoms.

[0029] All enrolled subjects underwent skin biopsy sampling according to standard procedures for the diagnostic assessment of intradermal nerve fiber density. 5 All participants signed a written informed consent form for participation in the study. Figure 1 The study is illustrated. Specifically, skin biopsy samples from two study groups (small fiber neuropathy (SFN) and healthy controls (HC)) were analyzed in two independent steps: an exploratory step and a validation step. In patients with small fiber neuropathy, two miRNAs were downregulated, and in SFN patients relative to HC, these two miRNAs exhibited synergistic effects. The remaining RNA samples (from 13 SFN patients and 10 HC patients) were used for gene transcription profiling as miRNA targets and to allow the identification of differentially expressed genes (DEGs) in SFN patients relative to the HC group. Experimental miRNA and mRNA data were then used to predict biological processes, gene ontology (GO) terms, and potential pathway alterations in the skin microenvironment of SFN disease on a computational basis. The functional miRNA-mRNA network of SFN patients was then reconstructed using experimental and computational data.

[0030] Skin biopsies and intraepidermal nerve fiber density (IENFD) assessment.

[0031] To evaluate IENFD, skin biopsies with a diameter of 3 mm were obtained from the distal end of the legs of all subjects, and were performed according to previously published protocols. 5The samples were then processed. In short, they were fixed in 2% paraformaldehyde-lysine-periodate solution at 4°C for 24 hours, cryoprotected overnight at 4°C, serially sectioned into 50 μm sections using a cryostat, and stored at -20°C until further processing. To evaluate IENFD, the 50 μm sections were immunostained with an antibody (1:500, Ultraclone) targeting protein gene product 9.5. IENFD was quantified using a Zeiss bright-field microscope, and according to previously published standards. 5,6 Perform the calculation.

[0032] RNA isolation from skin biopsy tissues According to the manufacturer's instructions, total RNA was isolated from the epidermis of two 50 μm skin biopsy sections from each subject under a microscope using the TruXtract FFPE Total RNA Kit (Covaris, catalog number PN520220) for the exploratory cohort and the PureLink™ FFPE Total RNA Isolation Kit (Invitrogen, catalog number K1560-02) for the validation cohort. Prior to preparing the miRNA array, the purity, concentration, and integrity of the RNA were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). All RNA samples meeting the required purity ratio (A260 / A280 = 1.7–2.0) were used for subsequent analysis.

[0033] Quantification According to the manufacturer's instructions, quantification of miRNA expression was performed using a pre-designed TaqMan™ array human microRNA A+B card (Thermo Fisher), containing 754 miRNAs, and pre-amplified. In short, 15 ng of total RNA was reverse transcribed, followed by pre-amplification of cDNA. The pre-amplified product was diluted in 75 μl of 0.1× TE buffer (pH 8.0) and used for RT-qPCR. The PCR reaction mixture was prepared using 9 μl of diluted pre-amplified product, 450 μl of TaqMan™ Rapid Advanced Premix, and 441 μl of nuclease-free water. Each slot was loaded with 100 μl of the PCR mixture and centrifuged. RT-qPCR experiments were performed on a ViiATM 7 Rapid Real-Time PCR system (Thermo Fisher Scientific). The following cycling protocol was used: enzymatic activation at 92 °C for 10 min, followed by 40 cycles, each cycle consisting of denaturation at 95 °C for 1 sec and annealing at 60 °C for 20 sec. The reaction volume for each microwell is 1 μl.

[0034] Quantification of gene expression Gene transcript expression was quantified using a custom-designed TaqMan™ microfluidic card array. Ninety-five priority gene transcripts were selected for their involvement in neuropathic pain-related pathways. Four subjects were analyzed simultaneously using each custom array. Residual RNA after miRNA analysis was reused and subjected to new quality controls. Twenty-three subjects (13 SFN patients and 10 HC patients) were analyzed, for whom we had sufficient samples. Reverse transcription was performed using the Invitrogen™ SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific), where 50 ng of total RNA was reverse transcribed into cDNA. All samples were then pre-amplified simultaneously. The pre-amplified PCR reaction was performed under the following thermal cycling conditions: enzyme activation at 95°C for 10 min, followed by 14 cycles, each consisting of denaturation at 95°C for 15 s and annealing at 60°C for 4 min. Enzyme inactivation was then performed at 99°C for 10 min. Samples were then diluted in 1×TE buffer (1:20 dilution factor) to prepare PCR mixtures. The wells were filled with 100 µl of the prepared mixture and run on a ViiATM 7 rapid real-time PCR system (Thermo Fisher Scientific) with the following thermal cycling program: enzyme activation at 95 °C for 10 min, followed by 40 cycles, each cycle consisting of denaturation at 95 °C for 15 sec and annealing at 60 °C for 1 sec. The reaction volume for each well was 1 µl.

[0035] Relative threshold method The "relative threshold" (Crt) method was used, which has been proven by the manufacturer to be the most robust method for analyzing data. 7 All raw PCR data were imported into DataConnect cloud in real time and automated CRT thresholds were applied in the design and analysis software (DA2) (ThermoFisher Scientific, online version). Therefore, only miRNAs with good amplification quality (Ampscore > 1 and Cqconf > 0.8) were included in the analysis.

[0036] NormFinder analysis Accurate determination of relative miRNA levels requires standardization using a reference or endogenous control. Ideally, this reference or endogenous control should be constant, stable, unregulated, and unaffected by experimental conditions. For this reason, its quality and stability in the target sample and study group must be considered when selecting it. In both the exploration and validation steps, NormFinder software was used to select endogenous miRNAs in two sample pools (A+B). In the exploration step, pool A was standardized using hsa-miR-200b-002251, and pool B was standardized using hsa-miR-1201-002781. In the validation step, hsa-miR-320-002277 (pool A) and hsa-miR-1247-002893 (pool B) were used as reference miRNAs. Non-human exogenous ath-miR159a was used as a negative control.

[0037] Relative expression analysis via 2-ΔΔct 8 Methods: Using HC tissue as the reference group, differential expression of miRNAs was quantified as relative quantification (QR). ΔCt was calculated as: the average ΔCt value of target miRNAs in the target group minus the average ΔCt value of target miRNAs in the reference group. Therefore, the fold change in expression was calculated as 2. -(ΔΔCq) .

[0038] In silico analysis of target genes To identify potential targets for differentially expressed miRNAs, a predictive analysis was performed on a computer by comprehensively searching information from miRTarBase, TargetScan, and Tarbase sources. Additionally, Cytoscape 3.9.1 was used. 9 The ClueGO app (v2.5.8) and DAVID 10 Software that identifies pathways and enriched biological processes from a list of target genes or a list of genes known to be involved in axonal degeneration and maintenance.

[0039] miRNAscope and immunofluorescence analysis MicroRNA in situ hybridization (ISH) was performed using the miRNAscope™ LS kit (Advanced Cell Diagnostics, Hayward, CA, USA) to locate miR-26b-5p in skin biopsy tissue. ISH was performed according to the kit instructions. In short, on day one, tissue adhesion was achieved according to the manufacturer's instructions, and a hydrophobic barrier was formed. After allowing the slide to dry overnight, ISH was performed on day two using the human-specific miRNA probe (miR-26b-5p). The fluorescent probe (DAPI, Invitrogen, catalog number D1306) was then incubated to identify the cell nucleus.

[0040] Statistical analysis The Wilcoxon-Mann-Whitney test was used to analyze the association between miRNA levels and clinical phenotypes (SFN and HC). The FDR Benjamini-Hochberg method was applied as correction for multiple tests. The Spearman correlation test was used to analyze the correlation between candidate miRNA expression and the expression of the putative target gene; a coefficient ≥ 0.5 or ≤ -0.5 with p < 0.05 was considered statistically significant. In the validation step, receiver operating characteristic (ROC) curves and area under the curve (AUC) were calculated to assess the ability of each miRNA to distinguish between SFN and HC patients. To explore the diagnostic accuracy of combining multiple miRNAs, combined ROC curves were calculated using multivariate logistic regression analysis.

[0041] Statistical analysis was performed using the statistical programming language R version 3.6 and STATA11 software.

[0042] Results Explore and validate novel candidate epidermal miRNAs associated with SFN. Study design and samples To explore the molecular profile of the epidermis in SFN patients compared to HC patients, a comprehensive analysis of miRNA and mRNA expression was performed. Figure 1 This study initially included 6 patients with idiopathic SFN and 7 patients with HC as a preliminary exploratory cohort, and analyzed them using TaqMan human microRNA cards containing 754 miRNAs. To further confirm the miRNA profiling results, an independent validation cohort of 11 SFN patients and 8 HC patients was included, using the same method. Furthermore, the mRNA expression of 95 gene targets involved in pathways related to neuropathic pain was analyzed in 13 SFN patients and 10 HC patients (whose biomaterials were still available).

[0043] The demographic and clinical characteristics of the study group are presented in Table 1.

[0044] There were no significant differences between SFN and HC patients in terms of sex (exploratory step p = 0.59, validation step p = 0.55) and age (exploratory step p = 1, validation step p = 0.20).

[0045] All subjects underwent skin biopsy for evaluation of IENFD, and patients showed a significant reduction in IENFD (Table 1 and ). Figure 2 A). This study included patients diagnosed solely with SFN.

[0046]

[0047] [“Età” = age;] "Sesso" = gender; “Distribuzione” = Distribution "Allodinia meccanica" = mechanical hyperalgesia; “iperalgesia da puntura di spillo” = Hypersensitivity to needle pricks; "Dolore (sintomi)" = pain (symptom); “Purito (sintomi)” = itching (symptom); “Evocato” = induce; "Iperalgesia termica" = thermal hyperalgesia; "Disfunzione vasomotoria / sudomotoria (segni)" = vasomotor / sweating dysfunction (signs); “Profilo sensoriale” = sensory spectrum; “Perdita sensoriale (segni)” = loss of sensation (signs); "Dolore profondo" = deep pain; "Freddo doloroso" = cold pain; "Bruciore dolorante" = burning pain; “Bruciante” = burning sensation; “Pungente parossistico” = paroxysmal stabbing pain Table 1. Demographic and clinical characteristics of the study population. Patients with SFN 1-SFN 6 were analyzed in the exploratory step, while patients with SFN 7-SFN 18 were included in the validation step. IENFD, intradermal nerve fiber density; LD, length-dependent pain distribution; N-LD, non-length-dependent pain distribution; NRS, numerical pain rating scale; sensory spectrum: TH, thermal hyperalgesia; MH, mechanical hyperalgesia; SL, sensory loss.

[0048] miRNA profiling analysis: exploratory step The microfluidic array contained 754 miRNAs, which were evaluated in all samples. After quality filtering, relative expression levels were used for further analysis. 11 Expression data for 111 miRNAs present in over 90% of samples were analyzed. In the epidermis of 6 SFN patients, compared to 7 HC subjects, 20 miRNAs with altered expression levels after multiple correction were observed, showing that 13 downregulated miRNAs and 7 upregulated miRNAs strongly distinguished phenotypes. Figure 2 B).

[0049] miRNA profiling analysis: validation step Then, in a separate cohort, skin biopsies from 12 SFN patients and 8 HC patients were validated using the same spectral analysis method (Table 1 and...). Figure 1 The study confirmed a significant difference in miR-26b-5p expression (adjusted p-value BH = 3.28E-02, fold change = -2.55), confirming the same downregulation pattern in SFN patients across both phases. Figure 3 A).

[0050] To investigate the relationship between miR-26b-5p expression and innervation, Spearman analysis was performed, which showed a strong correlation between miR-26b-5p and denervation level (calculated as the difference between measured IENFD and the cutoff value (δIENFD)), both in the exploratory step (r=0.77, p=0.0053) and the validation step (r=0.63, p=0.0055). Figure 3 BC).

[0051] Considering the synergistic effects among miRNAs, we extended our study to other uncorrected miRNA candidates to explore their potential relationships with the miR-26-5p candidate. Expression correlation analysis was performed, and a strong linear relationship was observed in the case of miR-20a-5p (p = 4.7e-07, R = 0.88). Figure 3D). In both the exploratory phase (HR = -1.48, unadjusted p = 4.80 E-02) and the validation phase (HR = -2.08, unadjusted p = 7.18 E-03), miR-20a-5p showed a downregulation trend in SFN patients. Figure 3 E), which also showed a strong correlation with δIENFD in both the exploratory step (r = 0.62, p = 0.037) and the verification step (r = 0.66, p = 0.0031). Figure 3 FG).

[0052] miR-26b-5p and miR-20a-3p in the epidermis showed a stronger discriminative power for SFN To test the ability of the two miRNA candidates to distinguish SFN, ROC analysis was performed and the area under the curve (AUC) was calculated. miR-26b-5p achieved an AUC of 96.4%, sensitivity of 91.7%, and specificity of 85.7%; while miR-20a-5p showed an AUC of 86.9%, sensitivity of 91.7%, and specificity of 81.4%. Figure 3 H).

[0053] Identification and validation of miRNA targets To further investigate the potential roles of miR-26b-5p and miR-20a-5p, a computer-based predictive analysis of target transcripts was performed by querying three databases (miRTarBase, Tarbase, and TargetScan). The identified genes were then used for biological pathway and process (BP) enrichment. Significantly enriched entries (p<0.05) were selected, which were considered to be closely related to neuronal development and maintenance, neuronal microenvironment homeostasis, and neurotrophic factor signaling.

[0054] Gene expression analysis was performed using a custom pain-related gene panel. Significantly abnormal expression of eight genes was observed (Table 2A): four downregulated (IKBKAP, MKNK2, PTGER3, and EDN1) and four upregulated (MEF2C, SLC25A36, NTRK2, and PIK3CG).

[0055] Table 2A

[0056] Gene expression analysis showed that the epidermal expression of eight genes was altered.

[0057] Paired Pearson correlation was used to identify the functional relationship between miRNA and target mRNA expression. Correlation analysis was applied to each miRNA and its putative target, which was significantly altered in SFN patients (Tables 2A and 2B). Figure 4). Our analysis showed that IKBKAP was correlated with the expression values ​​of miR-26b-5p (r=-0.75) and miR-20a-5p (r=-0.62); miR-26b-5p (r=0.75) and miR-20a-5p (r=0.67) were significantly correlated with MEF2C; both miRNAs were associated with MKNK2: miR-20a-5p (r=-0.52) and miR-26b-5p (r=-0.53); PIK3CG was associated with miR-20a-5p (r=0.53) and miR-26b-5p (r=0.53); and SLC25A36 was associated with miR-20a-5p (r=0.48) and miR-26b-5p (r=0.56), while miR-20a-5p expression was associated with NTRK2. (r=0.59) Related.

[0058] Table 2B

[0059] Gene annotation analysis To identify biological terms associated with aberrantly expressed genes, a gene annotation analysis was performed on a computer. Aberrantly expressed genes, corrected by Benjamini-Hochberg, were imported into the software, and, considering the tissues analyzed, the most relevant entries were selected from the associated categories (pathways and biological processes).

[0060] In situ hybridization of miR-26b Our miRNA profiling analysis provided a novel molecule that may play a regulatory role in the development of SFN; to investigate its cellular localization, miRNAscope in situ hybridization assays were performed, a known highly sensitive miRNA detection technique. In this experiment, we demonstrated that miR-26b-5p is highly concentrated in human epidermis. Figure 5 ).

[0061] In summary, the advantages of this invention will become immediately apparent.

[0062] In particular, miR-26b-5p and miR-20a-5p were confirmed to differentiate between patients with small fiber neuropathy and healthy subjects, with predictive power showing diagnostic accuracy of 96.4% and 86.9% respectively in ROC analysis.

[0063] A strong correlation was observed between innervation density (δIENFD) and miR-26b-5p or miR-20a-5p expression, supporting the hypothesis that miRNAs are involved in the maintenance of intradermal fibers, and suggesting that the significant downregulation of these two miRNAs is consistent with reduced cutaneous innervation.

[0064] The functions of these two miRNAs also provide an important foundation for the development of prevention strategies and drug treatments.

[0065] References 1.Devigili, G., Cazzato, D.&Lauria, G. Clinical diagnosis and management of small fiber neuropathy: an update on best practice. Expert RevNeurother 20, 967-980 (2020). https: / / doi.org:10.1080 / 14737175.2020.1794825 2.Wang, B.&Bao, L. Axonal microRNAs: localization, function and regulatory mechanism during axon development. J Mol Cell Biol 9, 82-90(2017). https: / / doi.org:10.1093 / jmcb / mjw050 3. Maimon, R. et al. miR126-5p Downregulation Facilitates AxonDegeneration and NMJ Disruption via a Non-Cell-Autonomous Mechanism in ALS. JNeurosci 38, 5478-5494 (2018). https: / / doi.org:10.1523 / JNEUROSCI.3037-17.2018 4.Chakraborty, C., Sharma, A. R., Sharma, G.&Lee, S. S. Therapeuticadvances of miRNAs: A preclinical and clinical update. J Adv Res 28, 127-138(2021). https: / / doi.org:10.1016 / j.jare.2020.08.012 5.Lauria, G. et al. Intraepidermal nerve fiber density at the distalleg: a worldwide normative reference study. J Peripher Nerv Syst 15, 202-207(2010). https: / / doi.org:10.1111 / j.1529-8027.2010.00271.x 6.Lauria, G.&Devigili, G. Skin biopsy as a diagnostic tool inperipheral neuropathy. Nat Clin Pract Neurol 3, 546-557 (2007). https: / / doi.org:10.1038 / ncpneuro0630 7.Biosystems, A. 8.Schmittgen, T. D.&Livak, K. J. Analyzing real-time PCR data by thecomparative C(T) method. Nat Protoc 3, 1101-1108 (2008). https: / / doi.org:10.1038 / nprot.2008.73 9.Shannon, P. et al. Cytoscape: a software environment for integratedmodels of biomolecular interaction networks. Genome Res 13, 2498-2504 (2003).https: / / doi.org:10.1101 / gr.1239303 10.Huang, d. W., Sherman, B. T.&Lempicki, R. A. Systematic andintegrative analysis of large gene lists using DAVID bioinformaticsresources. Nat Protoc 4, 44-57 (2009). https: / / doi.org:10.1038 / nprot.2008.211 11.Livak, K. J.&Schmittgen, T. D. Analysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method. Methods 25, 402-408 (2001). https: / / doi.org:10.1006 / meth.2001.1262。

Claims

1. The use of miRNA in diagnosing small fiber neuropathy (SFN) in patients, among which, The miRNA is one or both of the following: 。 2. The use of the miRNA according to the preceding claim in the diagnosis of small fiber neuropathy (SFN) in patients, wherein, One or two of the miRNAs were downregulated relative to healthy controls.

3. The use of the miRNA according to claim 1 or 2 in diagnosing small fiber neuropathy (SFN) in patients, wherein, The concentration of the miRNA was measured in isolated epidermal samples from the patient.

4. The medical applications of miRNA in treating patients with small fiber neuropathy (SFN), among which, The miRNA is one or both of the following: 。 5. The use of miRNA in the identification of therapeutic compounds for treating patients with small fiber neuropathy (SFN), wherein, The miRNA is one or both of the following: 。

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