Dysregulation of lncRNAs in autoimmune neuropathies

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and Guillain-Barré syndrome (GBS) are inflammatory neuropathies with different clinical courses but similar underlying mechanisms. Long non-coding RNAs (lncRNAs) might affect pathogenesis of these conditions. In the current project, we have selected HULC, PVT1, MEG3, SPRY4-IT1, LINC-ROR and DSCAM-AS1 lncRNAs to appraise their transcript levels in the circulation of CIDP and GBS cases versus controls. Expression of HULC was higher in CIDP patients compared with healthy persons (Ratio of mean expression (RME) = 7.62, SE = 0.72, P < 0.001). While expression of this lncRNA was not different between female CIDP cases and female controls, its expression was higher in male CIDP cases compared with male controls (RME = 13.50, SE = 0.98, P < 0.001). Similarly, expression of HULC was higher in total GBS cases compared with healthy persons (RME = 4.57, SE = 0.65, P < 0.001) and in male cases compared with male controls (RME = 5.48, SE = 0.82, P < 0.001). Similar pattern of expression was detected between total cases and total controls. PVT1 was up-regulated in CIDP cases compared with controls (RME = 3.04, SE = 0.51, P < 0.001) and in both male and female CIDP cases compared with sex-matched controls. Similarly, PVT1 was up-regulated in GBS cases compared with controls (RME = 2.99, SE = 0.55, P vale < 0.001) and in total patients compared with total controls (RME = 3.02, SE = 0.43, P < 0.001). Expression levels of DSCAM-AS1 and SPRY4-IT1 were higher in CIDP and GBS cases compared with healthy subjects and in both sexes compared with gender-matched healthy persons. Although LINC-ROR was up-regulated in total CIDP and total GBS cases compared with controls, in sex-based comparisons, it was only up-regulated in male CIDP cases compared with male controls (RME = 3.06, P = 0.03). Finally, expression of MEG3 was up-regulated in all subgroups of patients versus controls except for male GBS controls. SPRY4-IT could differentiate CIDP cases from controls with AUC = 0.84, sensitivity = 0.63 and specificity = 0.97. AUC values of DSCAM-AS1, MEG3, HULC, PVT1 and LINC-ROR were 0.80, 0.75, 0.74, 0.73 and 0.72, respectively. In differentiation between GBS cases and controls, SPRY4-IT and DSCAM-AS1 has the AUC value of 0.8. None of lncRNAs could appropriately differentiate between CIDP and GBS cases. Combination of all lncRNAs could not significantly enhance the diagnostic power. Taken together, these lncRNAs might be involved in the development of CIDP or GBS.


Scientific Reports
| (2021) 11:16061 | https://doi.org/10.1038/s41598-021-95466-w www.nature.com/scientificreports/ levels in the circulation of CIDP and GBS cases versus controls. The reason for selection of these lncRNAs was their roles in modulation of immune responses. HULC has been identified as one of important factors in induction of pro-inflammatory responses in the course of liposaccharide-associated sepsis in endothelial cells 10 . Pvt1 has been shown to modulate the immunosuppression function of granulocytic myeloid-derived suppressor cells in animal models 11 . MEG3 has been reported to induce imbalance between regulatory T cells and Th17 cells 12 . SPRY4-IT1 interacts with ERRα 13 , a nuclear receptor which regulates innate immunity 14 . LINC-ROR has functional interaction with TGF-β to regulated hypoxia-induced cellular cascades 15 . Finally, DSCAM-AS1 has been shown to regulate several genes which are implicated in inflammatory responses 16 . These lncRNAs regulate immune reactions via different routes.

Materials and methods
Recruitment of GBS/CIDP cases and normal controls. A total of 32 CIDP patients with typical type (11 females, 21 males), 25 GBS patients (7 females, 18 males), and 58 healthy individuals (20 females and 38 males) participated in the current investigation. CIDP cases had symmetric muscle weakness which affected both proximal and distal muscles. The course of disorder was compliant with a motor-predominant neuropathy. Patients were assessed using the guidelines stated by American Academy of Neurology 17 and National Institute of Neurological Disorders and Stroke 18 . In addition, electrophysiological criteria were used for diagnosis of GBS 19 . Blood samples were obtained when patients entered the remission phase and were not on any treatment. All were responsive to corticosteroids or IVIg treatment. Ethics approval and consent to participant. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent forms were obtained from all study participants. The study protocol was approved by the ethical com-  Table 2 demonstrates demographic and clinical data of patients. Expression of HULC was higher in CIDP patients compared with controls (Ratio of mean expression (RME) = 7.62, SE = 0.72, P < 0.001). While expression of this lncRNA was similar between female CIDP  www.nature.com/scientificreports/ cases and female controls, its expression was up-regulated in male CIDP cases compared with male controls (RME = 13.50, SE = 0.98, P < 0.001). Similarly, expression of HULC was higher in total GBS cases compared with controls (RME = 4.57, SE = 0.65, P < 0.001) and in male cases compared with male controls (RME = 5.48, SE = 0.82, P < 0.001). Similar pattern of expression was detected between total cases and total controls. PVT1 was up-regulated in CIDP cases compared with controls (RME = 3.04, SE = 0.51, P < 0.001) and in both male and female CIDP cases compared with sex-matched healthy persons. Similarly, PVT1 was up-regulated in GBS cases compared with controls (RME = 2.99, SE = 0.55, P vale < 0.001) and in total patients compared with total controls (RME = 3.02, SE = 0.43, P < 0.001). Expression levels of DSCAM-AS1 and SPRY4-IT1 were higher in CIDP and GBS cases compared with controls and in both sexes compared with gender-matched healthy subjects. Although LINC-ROR was up-regulated in total CIDP and total GBS cases compared with controls, in sex-based comparisons, it was only up-regulated in male CIDP cases compared with male controls (RME = 3.06, P = 0.03). Finally, expression of MEG3 was up-regulated in all subgroups of patients versus controls except for male GBS controls (Table 3). Figure 1 displays expression amounts of selected lncRNAs in study subgroups. Significant pairwise correlations have been identified between lncRNAs expressions with the most robust one being between HULC/DSCAM-AS1 and HULC/SPRY4-IT pairs (r = 0.86 and 0.85 respectively) (Fig. 2).
Finally, diagnostic power of lncRNAs for distinguishing patients from healthy subjects was assessed (Fig. 4). SPRY4-IT could differentiate CIDP cases from controls with AUC = 0.84, sensitivity = 0.63 and specificity = 0.97. AUC values of DSCAM-AS1, MEG3, HULC, PVT1 and LINC-ROR were 0.80, 0.75, 0.74, 0.73 and 0.72, respectively. In differentiation between GBS cases and controls, SPRY4-IT and DSCAM-AS1 has the AUC value of 0.8. None of lncRNAs could appropriately differentiate between CIDP and GBS cases. Combination of all lncRNAs could not significantly enhance the diagnostic power (Table 4).

Discussion
LncRNAs have been shown to take part in the pathogenesis of immune-related conditions. Up-regulation of lncRNAs has been reported in a number of these conditions. For instance, expression levels of HOTAIR, LUST, anti-NOS2A, MEG9, SNHG4, TUG1, and NEAT1 have been shown to be increased in blood exosomes of patients with rheumatoid arthritis (RA) compared with exosomes retrieved from normal blood samples 21 . The same study has reported up-regulation of mentioned lncRNAs in addition to H19 antisense, HAR1B and GAS5 in peripheral blood mononuclear cells of these patients 21 . ENST00000483588 is another lncRNA which has been shown to be up-regulated in fibroblast-like synoviocytes of patients with RA 22 . A number of selected lncRNAs in the current www.nature.com/scientificreports/ project have been previously shown to be up-regulated in immune-mediated conditions. For instance, PVT1 has been reported to be up-regulated in fibroblast-like synoviocytes of RA models parallel with down-regulation of sirt6, a putative target for this lncRNA. PVT1 silencing or sirt6 over-expression could suppress cell proliferation and inflammation, while inducing cell apoptosis 23 . MEG3 has been demonstrated to regulate RA pathogenesis through targeting NLRC5 24 . LINC-ROR, MEG3, SPRY4-IT1 and UCA1 have been among lncRNA with higher expression in patients with schizophrenia compared with normal subjects 25 . CIDP and GBS disorders are two immune-mediated conditions in which lncRNAs might contribute. We measured expression of amounts of six immune-related lncRNAs in the circulation of these patients versus healthy controls. Expression of HULC was higher in CIDP patients compared with controls. While expression of this lncRNA was not different between female CIDP cases and female controls, its expression was higher in male CIDP cases compared with male controls. Similarly, expression of HULC was higher in total GBS cases compared with controls and in male cases compared with male controls. Similar pattern of expression was detected between total cases and total controls. HULC has been shown to regulate immune responses through miR-128-3p/RAC1 axis 26 . In line with our observations, miR-128-3p has been shown to be down-regulated in cerebrospinal fluid of animal models of GBS 27 . RAC1 regulates a number of inflammatory pathways such as STAT3 and NF-κB 28 . NF-κB pathway has a documented effect in the pathogenesis of immune-related neuropathies 29 . Therefore, HULC/miR-128-3p/RAC1 axis might also been involved in the pathogenesis of CIDP and GBS.
PVT1 was up-regulated in CIDP cases compared with controls and in both male and female CIDP cases compared with sex-matched controls. Similarly, PVT1 was up-regulated in GBS cases compared with controls and in total patients compared with total controls. Contrary to this finding, we have previously reported downregulation of PVT1 in the peripheral blood of patients with multiple sclerosis 30 . Therefore, this lncRNA might have distinctive effects in these two inflammatory conditions. Expression levels of DSCAM-AS1 and SPRY4-IT1 were higher in CIDP and GBS cases compared with controls and in both sexes compared with sex-matched controls. Therefore, these lncRNAs have a consistent pattern of expression among CIDP and GBs patients potentiating them as biomarkers for these conditions.
Although LINC-ROR was up-regulated in total CIDP and total GBS cases compared with controls, in sexbased comparisons, it was only up-regulated in male CIDP cases compared with male controls indicating the www.nature.com/scientificreports/ possible interactions between this lncRNA and sex-related parameters, since there was no gender-based difference in phenotype of the patients in terms of severity of illness. Finally, expression of MEG3 was up-regulated in all subgroups of patients versus controls except for male GBS controls. Expression of MEG3 has been shown to be elevated in CD4 + T cells of patients with immune thrombocytopenic purpura. Expression of this lncRNA has been reduced in CD4 + T cells cultured with dexamethasone 12 . Functionally, MEG3 inhibits Foxp3 expression and increases RORγt expression, thus inducing imbalance between regulatory T cells and Th17 cells 12 . The imbalance between these subsets of T cells might participate in the pathogenesis of GBS or CIDP as previous studies have shown the therapeutic effects of regulatory T cells in animal models of GBS 31 .
The correlations between expression levels of mentioned lncRNAs were not meaningfully different between patients and controls based on the measured correlation coefficients. SPRY4-IT and DSCAM-AS1 could differentiate CIDP cases from controls with appropriate diagnostic power values. Similarly, these lncRNAs had high power in differentiation between GBS cases and controls. Since expression levels of lncRNAs were almost similar between CIDP cases and GBS cases, none of lncRNAs could appropriately differentiate between CIDP and GBS cases. Combination of all lncRNAs could not significantly enhance the diagnostic power. Taken together, these lncRNAs might be involved in the development of CIDP or GBS. These transcripts might be regarded as marker for these immune-related conditions as well. Future studies should appraise expression of these transcripts in other immune-related conditions to evaluate their suitability as diagnostic markers for GBS/CIDP.   www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.