Expression and diagnostic values of MIAT, H19, and NRON long non-coding RNAs in multiple sclerosis patients

Multiple sclerosis (MS) is a chronic inflammatory disease. Various long non-coding RNAs (lncRNAs) appear to have an important role in the pathophysiology of MS. This study aimed at evaluating the expression levels of lncRNAs, MIAT, H19, and NRON in peripheral blood of MS cases to a healthy control group. We collected blood samples of 95 MS cases (76 relapsing–remitting (RR) and 19 secondary progressive (SP) MS) and 95 controls. We used quantitative real-time PCR for the evaluation of gene expression. The correlation between expression with clinical parameters was analyzed by a multiple linear regression model. Receiver operating characteristic (ROC) curve analysis was carried out to detect the diagnostic potential of lncRNAs levels according to the area under the curve (AUC). MIAT, H19, and NRON were significantly increased in the RRMS and SPMS subgroups compared to the controls. We found that the H19 and MIAT expression significantly were higher in SPMS compared with RRMS. Patients with RRMS had a greater level of the average NRON expression is compared with SPMS patients. The expression level of H19 significantly was higher in females relative to male patients. Based on the area under curve (AUC) values, NRON had the best performance in the differentiation of MS patients from controls (AUC = 0.95, P < 0.0001). A combination of MIAT, H19, and NRON expression levels could be useful in differentiating MS patients with 93.6% sensitivity, 98.9% specificity, and diagnostic power of 0.96 (P < 0.0001). The levels of MIAT, H19, and NRON in peripheral blood could be important biomarkers for MS diagnosis.


Background
Multiple sclerosis (MS) is a chronic complex autoimmune disease that causes the demyelination of neurons in the central nervous system (CNS) [1]. MS is especially common in young female adults [2]. A broad range of symptoms may appear in MS patients, such as pain, visual sensory disturbance, motor impairments, cognitive deficits, and fatigue. The different factors such as autoimmunity, genetic predisposition, and environmental influenced the pathophysiology of MS diseases. Evidence indicates that the reaction between abnormal T cells and CNS autoantigens can cause inflammation, demyelination, and neurodegeneration [3]. In addition, B cells act as an important source of plasma cells that generate antibodies while also regulating T cell production and autoimmune processes. B cells may affect the pro-inflammatory process of other immune cells [4].
Many genomic studies have demonstrated the presence of a large portion of DNA that doesn't code for proteins, but is transcribed to the different types of RNAs. The lncRNAs are a type of non-coding RNAs known to possess the ability to regulate cell proliferation through different mechanisms [5]. The dysregulation of lncRNAs is considered in the cause of many neurological disorders [6][7][8]. Research works have indicated that the expression of lncRNAs is significantly dysregulated in MS patients [9][10][11][12][13]. Immune regulation was affected by the function of different lncRNAs. A previous study revealed an important connection between the lncRNA MIAT (Myocardial infarction-associated transcript) and inflammatory response [14]. MIAT regulates the differentiation of neural stem cells into oligodendrocytes in the CNS [15]. The NRON (Non-Coding Repressor Of NFAT) lncRNA is an endogenous ribonucleic acid that is detected in human T cells and can regulate IL-2 expression and NFAT Ca 2+ -activated transcription factor in activated T cells [16].
The lncRNA H19 is a lncRNA with a critical role in immune response regulation. It is also an imprinted gene that is expressed only from the maternal allele [17,18].
Although the role of MIAT, H19, and NRON in cellular pathways and different cancers has been assessed, only one study has been conducted to evaluate the expression level of NRON lncRNA in 10 MS cases. Because there is evidence showing that lncRNAs are stable in human serum, circulating serum RNA levels could serve as a biomarker in non-invasive diagnostic applications [19,20].
Research on the role of lncRNAs in MS pathogenesis is just beginning. The significance of the present study becomes more apparent due to high incidence and prevalence of MS in Iran [21]. Therefore, we aimed to assess the effectiveness of using known lncRNAs as potential biomarkers for the diagnosis of MS.

Study subjects
The present case and control study was carried out to assess the expression levels of three lncRNAs (MIAT, H19, and NRON) in 95 MS cases, who were compared to 95 controls from August 2018 to August 2020 at Namazi Hospital in Shiraz. Among all patients, 76 individuals were relapsing-remitting (RR) and 19 were secondary progressive (SP). Controls comprised a representative sample of Shiraz's population; sex-and age-matched controls were randomly selected from neighbor controls at the nearest case's place matched.
All participants in the present study were clinically diagnosed by an expert neurologist according to McDonald's criteria [22]. All cases included in this study showed EDSS progression with no evidence of a relapse in the last 24 months. In the present study, we considered the following characteristics as exclusion criteria: recent infection, smoking history, malignancy, alcohol abuse, and the presence of any other inflammatory or autoimmune diseases. The examined subjects were aged 20-56 years.
Ethics approval for this research was obtained by the local ethics committee of the Kazeroon Branch, Islamic Azad University, Iran (IR.IAU.KAU.REC.1398.034). The subjects completed a written informed consent and voluntarily agreed to participate in the present study as a part of a large prospective project.

Collection of blood samples
After obtaining an informed consent form, the peripheral venous blood samples were collected from MS cases and control individuals in ethylene diamine tetra acetic acidcoated (EDTA) tubes as an anticoagulant.

RNA extraction and cDNA synthesis
A total RNA extraction kit (Favorgen, Taiwan) was used to isolate Total RNA from peripheral blood according to the manufacturer's instructions. cDNA synthesis Kit (Yektatajhiz, Iran) was employed for cDNA synthesis from RNA samples. For cDNA synthesis, RNA samples with theA260/A230 and A260/A280 ratios greater than 1.7 were selected.

Quantitative RT-PCR measurement of the lncRNA levels
Primer Express and Gene Runner software v.3.0 were applied to design and analyze the oligonucleotide primers. By searching the BLAST website, the primers were confirmed to avoid non-specific PCR product formation. Specific primers' evaluations of MIAT, H19, NRON, and the TBP gene (used as a reference gene) are shown in Table 1. Gene expression was normalized relative to the housekeeping genes GAPDH and TBP. TBP was selected as the preferential reference gene given its low variance in Ct between the different samples (data not shown).  [23].

Statistical analysis
The IncRNAs relative expression levels were reported as mean ± SE. Student's t test was employed to compare the mean expression of each lncRNA gene between case and control groups. The Beta coefficient of lncRNAs expression levels was estimated in univariate and multiple linear regression analyses. Using a multiple linear regression model, we adjusted for the most important clinical factors. Receiver operating characteristic (ROC) curve analysis was carried out to detect lncRNAs levels as a diagnostic biomarker for MS according to the area under the curve (AUC). We used the multiple logistic regressions to combine the age and sex variables in ROC analysis. The analyses were performed using the Graph-Pad Prism 5.0 and the SPSS software (version 19.0). The P value of < 0.05 was regarded as statistically significant.

Clinical and demographic characteristics
Clinical and demographic characteristics of MS cases and healthy controls are summarized in Table 2. No significant difference was demonstrated in sex and age ratio (P > 0.05). Figure 1 demonstrates that MIAT, H19, and NRON levels were significantly higher in MS cases than in healthy controls (P < 0.0001), the median log 2 fold changes were as follows, respectively 1.10, 2.79, and 1.30. The significantly high expression of MIAT, H19, and NRON can distinguish MS cases from healthy cases (Fig. 1A). Then, lncRNAs expression levels were compared in RRMS and SPMS subtypes with healthy controls separately. MIAT, H19, and NRON expression levels were significantly increased in the RRMS and SPMS subgroups when compared to the controls separately (P < 0.001). Oneway ANOVA analysis showed that the MIAT and H19 expression in SPMS significantly was higher than RRMS, while NRON level in RRMS was higher relative to SPMS (Fig. 1B).

Association between clinical variables and LncRNAs expression
Multiple linear regression analysis was done to detect the association between RRMS and SPMS patients and lncR-NAs expression levels after adjusting for several clinical confounders (

Predictive power of MIAT, H19, and NRON in MS diagnosis
ROC curve analyses were done to assess the diagnostic values of MIAT, H19, NRON, and whole lncRNAs expression levels and their usefulness in predicting MS. The results of these analyses are presented in Fig. 2 and Table 4. Three lncRNAs were shown to be effective in differentiating MS cases from controls. MIAT had an AUC of 0.861 (95% CI: 0.804-0.918;

Discussion
The present case-control study was performed on southwest Iranian individuals. We reported an upregulation of MIAT, H19, and NRON lncRNAs in the peripheral blood of MS patients in comparison with healthy subjects, the median log 2 fold changes were as follows, respectively, 1.10, 2.79, and 1.30. The previous studies reported the dysregulation of various lncR-NAs in MS cases [9][10][11]. For example, Shaker et al. found a significant increase in the expression levels of lnc-DC and MALAT1 in MS patients [10]. Dastmalchi et al. showed that NEAT1, PANDA, and TUG1 lncR-NAs were over-expressed in MS cases when compared with controls [11]. One previous study [13] showed that three lncRNAs (NEAT1, TUG1, and RN7SKRNA) were up-regulated in RR patients. The downregulation and upregulation of six lncRNAs in MS cases were investigated in another study [24]. We found that the expression levels of MIAT and H19 in SP patients were significantly higher than in RR patients. However, NRON expression levels in RR patients showed significantly higher expression relative to SP patients. NRON is a lncRNA repressor that acts as a specific regulator of NFAT nuclear trafficking and interacts with the importin-beta superfamily [25]. NFATs regulate the transcriptional induction of genes that encode for immune activators/modulators such as GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-13, L-8, IFNγ,CD5, CD25, CD28, and CD40 [26]. In T cells, NFAT proteins govern gene expression, thereby regulating their activation, differentiation, and development in addition to the maintenance and induction of T cell tolerance [27]. The expression level of the NRON lncRNA was significantly higher in MS patients than in controls. This outcome is contrary to that of Fenoglio et al., who found that NRON was downregulated in peripheral blood of 10 MS cases in comparison with controls. They showed that NRON correlates with MS duration [12].
In Fenoglio study, the downregulation of NRON in 10 cases (5 RRMS and 5 PPMS) was evaluated, while in our study, lncRNAs expression was evaluated in 95 MS patients (76 RRMS and 19 SPMS). We also reported the significant upregulation of NRON in two subtypes of MS relative to control. Additionally, our results showed no significant correlation between the expression levels of NRON with disease duration, EDSS score, and age among MS patients. The mean disease duration in our study was 7.82 ± 6.00 years it seems that the difference in duration of the MS disease in the two studies has led to this controversy. NRON expression is likely to be high in the early years of the MS disease and then gradually decreases.
The H19 lncRNA has an important role in the regulation of immune response [17]. H19 is an imprinted gene that is expressed from its maternal allele [18]. The deletion of H19 genes facilitates cell cycle progression in hematopoietic stem cells through the induction of maternal IGF2 [28]. H19 might contribute to the pathogenesis of MS based on its role in the IGF system and the importance of hematopoietic stem cells in the regulation of immune response [29,30]. One previous investigation demonstrated the participation of H19 in rheumatoid arthritis (RA) pathology according to its overexpression in RA cases and its hypersensitivity to cytokine regulation/starvation in these cases [31]. The upregulation of H19 has been reported in ischemic stroke patients with a potential for the early diagnosis of IS [32]. Zeis et al. demonstrated that over-expression of the IGF1 and IGF2 genes was found in inactive demyelinated lesions [33]. We demonstrated that the expression level of H19 was significantly up-regulated in the blood of MS cases in comparison with controls. We also observed that H19 expression levels correlated with sex so that the higher expression in females was detected compared with male patients. The female-specific elevated expression of H19 was reported in a previous study [34]. Adriaenssens et al. reported that 17-beta-estradiol stimulated the endogenous H19 gene in MCF-7 cells [35]. On the other hand, the estrogen-ERα-H19 signaling axis may also be important in the development of breast cancer [36]. This gene acts as an estrogen receptor modulator [37]. As many articles have suggested the importance of sex hormones in determining the onset and outcome of multiple sclerosisin in females [38,39]. These findings have raised the challenging question of whether estrogen may be present in the pathophysiology of MS disease by overexpression in H19 lncRNA? Further studies with a larger sample size are needed to confirm the strong correlation between sex and H19 expression level in MS patients. Our results also showed that the H19 expression in SPMS patients was 5.56 times higher than that in RRMS patients. SPMS patients are usually older than RRMS with higher disability and hospitalization rates relative to RR [40]. But in our study, patients with SPMS (n = 19) had not have higher EDSS scores and age compared with RRMS (76). Thus, we can suppose that a larger sample size for SPMS patients is needed to clarify the significant difference between age and EDSS with RRMS. This result indirectly suggests that H19 expression in MS disease may have a positive correlation with the disability score of patients but larger sample size is needed to reach this correlation.
MIAT is a well-characterized lncRNA that affects cellular functions such as apoptosis, invasion, and proliferation in many human diseases. The regulatory mechanism of MIAT is very complex [41]. In patients with coronary artery disease (CAD) compared with controls, the serum level of MIAT was significantly increased and positively correlated with serum IL-6 and TNF-α [42]. Previous research works have shown that the MIAT level can be used as a biomarker for the diagnosis and prognosis of different diseases such as CAD [42,43], various cancers [44,45], and ischemic stroke [46]. Zhu et al. demonstrated that the expression levels of MIAT were significantly up-regulated in the leukocytes of ischemic stroke (IS) patients in comparison with controls [46]. We demonstrated that the expression level of MIAT was significantly higher in MS cases than in controls. Also, in the present study, we showed that MIAT expression was 5.93 times higher in SP cases than in RR cases. We could not detect the positive correlation between MIAT expression and EDSS score but significantly high expression of MIAT in SPMS relative to RRMS indirectly may show the positive association between MIAT and MS severity.
A valuable method for the early screening of MS is developing a high-sensitive non-invasive blood biomarker. Evidence shows that circulating lncRNAs could be considered MS biomarkers [10,11,47]. Elevated MIAT, H19, and NRON expression levels have been reported as characteristics of various disorders (48)(49)(50)(51)(52)(53). Therefore, they could also be used as a non-specific biomarker for MS. Among the three lncRNAs investigated in the present study, NRON showed the best efficiency as a biomarker-based on its sensitivity, specificity, and AUC values.
In the present study, all patients were chosen consecutively from hospitals during the same period. The participants represented a variety of ethnic groups, though selection bias could not be avoided. More accurate findings could be observed with larger sample sizes to verify our results. Also, in the current investigation, we evaluated the expression levels of lncRNAs without performing a full transcriptome analysis. Furthermore, more investigations are needed to detect the exact molecular mechanisms by which MIAT, H19, and NRON participate in MS pathology. Although the current study indicates that circulating MIAT, H19, and NRON can be used as diagnostic biomarkers for MS, further research is required to find reliable blood biomarkers for clinical use.

Conclusions
This research showed the upregulation of three specific lncRNAs (MIAT, H19, and NRON) in the peripheral blood of 95 MS cases. Additional research with larger sample size is needed to confirm the present findings and clarify the molecular mechanisms of these lncRNAs in the pathogenesis of MS. Their dysregulation profiles indicate that they could be considered potential biomarkers for predicting the course of MS or patients' responses to treatment.