Emerging roles of very large (> 5 kb) lncRNAs in immune regulation and disease processes are being discovered [
11]. Here we report the expression signature of ten lncRNAs NTT, NEAT1, MALAT1, TUG1, MIAT, His-1 RNA, GNAS1-AS, EMX2OS, CR933609 and AK124742 in PBMCs derived from ME/CFS patients. We have selected this screening panel according to their potential regulation of immune, stress, metabolic and neurologic responses, which have been hypothesized to be involved in ME/CFS pathogenesis. Nevertheless it is remarkable to find out on PCA plot that ME/CFS could be differentiated from healthy controls via this lncRNA profile. Among the ten lncRNAs, NTT, MIAT and EMX2OS expression levels explained the most variance between ME/CFS and controls. Further supporting their potential role in disease pathomechanism, higher NTT and EMX2OS levels were associated with more severe ME/CFS (Bell score < 30). Using expression of any two of these three lncRNAs (NTT, MIAT and EMX2OS) in discriminating ME/CFS from healthy had an AUC of 0.82 on ROC curve, suggesting a potential diagnostic value of these lncRNAs for ME/CFS.
Consistent with the hypothesis that disease pathology in ME/CFS could be driven by oxidative stress and viral infections, we found that NTT and MIAT levels in THP-1 and KALS1 cell lines could be increased after H
2O
2 or poly (I:C) stimulations, an expression pattern similar to those found in ME/CFS patients’ PBMCs. However, we do not know yet whether this lncRNA profile found in our study is specific for ME/CFS or can be found in other diseases involving immune dysregulation or oxidative stress, such as autoimmune diseases and cancer. It has been reported that MIAT levels could be upregulated in high glucose conditions and in lung cancer, and NTT expressions might be found in processes involving T cell activation [
12,
17,
29]. Further comparing the PBMC expression signature of NTT, MIAT, and EMX2OS in ME/CFS with patients suffering from chronic fatigue due to autoimmune diseases or cancer is important to assess the lncRNA test specificity in diagnosing ME/CFS.
The mechanisms of NTT, MIAT and EMX2OS in ME/CFS pathogenesis require further investigations. In our study, we detected an association of higher level of
ZEB1, a MIAT-regulated gene, with ME/CFS. Consistently, both THP-1 and KALS1 cell lines showed higher expression levels of MIAT and
ZEB1 after stimulation of poly (I:C), a synthetic analog of double strand RNA representing active viral infection, a potential trigger of ME/CFS. Zinc finger E-box-binding protein (ZEB) 1 has been reported to be a transcription factor recruiting repressor complex to suppress IL-2 activation in T cells [
30]. Upregulation of
ZEB1 might be associated with response to chronic inflammation in ME/CFS. In non-small-cell lung cancer cell line, knockdown of MIAT resulted in decreased
ZEB1 expression, indicating cis-action of MIAT on regulating
ZEB1 [
27]. Interestingly MIAT is involved in endothelial dysfunction as well, which is frequently observed in ME/CFS [
17,
31]. For NTT, it has been proposed to exert its function on nearby genes due to its large size (17 kb) [
12]. Several genes involving cell proliferation, apoptosis or inflammation, including
IFNGR1,
PBOV1,
TNFAIP3,
HIVEP2,
BCLAF1 and
MYB are located close to the chromosome position of NTT [
12]. We found no significant difference in
IFNGR1 and
PBOV1 expressions between ME/CFS and controls. However, a marked positive correlation between NTT and
IFNGR1 levels were observed in ME/CFS, not in controls. This observation suggests that the NTT/
IFNGR1 axis might play a subtle role in ME/CFS pathogenesis. Whether other downstream genes are affected by the upregulated NTT in ME/CFS and possibly play more important roles in disease pathobiology needs more experiments. Finally, according to lncrnadb, the lncRNA database, the expression level of EMX2OS under normal physiological condition is higher in brain, moderate in lymph nodes, and very low in leukocytes. Consistent with this, we could not detect EMX2OS in PBMCs of several individuals. However, most ME/CFS patients were found to have elevated EMX2OS in their PBMCs. The role of EMX2OS in PBMC is currently unclear, and the potential downstream gene
EMX2 usually expressed in CNS could not be detected in all study subjects [
22]. Further EMX2OS overexpression experiment in THP-1 is underway in our lab to answer this question. Interestingly, E
MX2 upregulation was found in brain hypoxemia [
32]. ME/CFS patients have broad decreases in cerebral blood flow, which may result in hypoxemia [
33].
In addition to our study on using lncRNA signature as diagnostic marker for ME/CFS, profiles of blood mRNA expression and plasma metabolites have been suggested to show diagnostic values for ME/CFS [
6,
7,
34]. As described by Kerr et al., there were 88 differentially expressed genes in ME/CFS required for diagnostic and prognostic grouping [
34]. Furthermore, Naviaux et al. proposed an eight-metabolite set for diagnosing male ME/CFS, and a thirteen-metabolite set for diagnosing female ME/CFS [
6]. Our results showed that a lncRNA expression panel composed of as few as three very large lncRNAs (NTT, MIAT, EMX2OS) may achieve a good diagnostic value and provide information about ME/CFS disease severity (NTT, MIAT).