PBMC lncRNA profile in chronic fatigue patients and controls

Expression of the ten lncRNAs (NTT, NEAT1, MALAT1, TUG1, MIAT, His-1 RNA, GNAS1-AS, EMX2OS, CR933609 and AK124742) in peripheral blood mononuclear cells (PBMCs) of ME/CFS patients and healthy controls were evaluated by RT-PCR. The values of the ten ΔCT (CTlncRNA–CTGAPDH) of each individual subject were used to build a correlation matrix, and projected to principle component space by principle component analysis (PCA) (Fig. 1a). The PCA showed that the ME/CFS and the control lncRNA expression profile could be separated mainly by principle component 2 (dimension 2). Similarly, the eigenvalues showed that data variances is mostly explained by the first and the second components (Fig. 1b).

Fig. 1 Principle component analysis (PCA) of ΔCT values of ten lncRNAs from PBMCs of ME/CFS patients and controls. a Variance between ME/CFS (red dots) and control (green dots) according to the ten lncRNA correlation matrix, displayed onto a two-dimension plot. b Eigenvalues of principle components Full size image

Association of lncRNA expression levels with ME/CFS and disease severity

In order to reduce the test numbers of lncRNAs needed for differentiating ME/CFS from healthy controls, and to evaluate the association of lncRNA expression level with ME/CFS disease severity, we analyzed the amount of each lncRNA in PBMCs of controls, mild-moderate ME/CFS (with Bell score ≥ 30), and severe ME/CFS (with Bell score < 30) (Fig. 2a–j). Interestingly, NTT, MIAT and EMX2OS levels were found to be significantly higher in ME/CFS PBMCs as compared with controls (ME/CFS median ΔCT vs. control median ΔCT: NTT 8.86 vs. 10.05, Mann–Whitney U test p < 0.0001; MIAT 6.22 vs. 6.89, Mann–Whitney U test p < 0.05; EMX2OS 20.69 vs. 18.59, Mann–Whitney U test p < 0.001). Furthermore, NTT and EMX2OS expression levels correlated with ME/CFS disease severity, with highest amount detected in ME/CFS patients with Bell score of 10 or 20 (< 30) (Fig. 2a, h).

Fig. 2 Expression of the ten very long lncRNA in PBMCs of ME/CFS and controls (a–j). ME/CFS patients with Bell score < 30 belong to the severe group. *p < 0.05, **p < 0.01, ***p < 0.001 by Mann–Whitney U tests. Lines represent medians. ns not statistically significant Full size image

PBMC NTT, MIAT and EMX2OS expression signature as ME/CFS diagnostic markers

Since we have discovered that among the ten lncRNAs tested, only NTT, MIAT and EMX2OS levels were upregulated in ME/CFS, we plotted a new PCA plot using a correlation matrix of NTT, MIAT and EMX2OS ΔCT values (Fig. 3a). In Fig. 3a, ME/CFS patients could be separated from healthy controls by principle component 1 (dimension 1). The ME/CFS group could still be separated from the control group when the input lncRNA values were reduced to those of NTT and MIAT (Fig. 3b) or those of NTT and EMX2OS (Fig. 3c). However, the ME/CFS and the control group could not be well differentiated from each other when the PCA was performed on a correlation matrix of only MIAT and EMX2OS (Fig. 3d).

Fig. 3 Diagnostic value of NTT, MIAT and EMX2OS expressions in ME/CFS. PCA showing the distribution of ME/CFS and controls on two-dimension plots differentiated by values derived from the expression levels of NTT, MIAT and EMX2OS (a) or from the expression levels of any two of the three lncRNAs (b–d). e Receiver-operating characteristic (ROC) curve analysis of the three-lncRNA signature to discriminate ME/CFS patients from healthy controls. AUC = 0.82 when the criteria is increased expression (ΔCT below the optimal cutoff) of any two of the three lncRNAs (NTT, MIAT and EMX2OS) Full size image

To evaluate the diagnostic value of the combination of the expression of NTT, MIAT and EMX2OS, we first obtained the optimal cutoff ΔCT value of each of the three lncRNAs by using receiver operating characteristic (ROC) curves. The optimal cutoff ΔCT value for NTT, MIAT and EMX2OS were 9.49 (area under the curve (AUC) = 0.82, 95% CI 0.72–0.90); 6.82 (AUC = 0.65, 95% CI 0.53–0.75); 19.06 (AUC = 0.77, 95% CI 0.64–0.87), respectively. Further ROC analysis was then performed and revealed that the criteria of increased expression (ΔCT below the optimal cutoff) of any two of these three lncRNAs in diagnosing ME/CFS had a sensitivity of 67.4, and specificity of 86.7, AUC = 0.82, with 95% CI 0.71–0.90 (Fig. 3e).

Expression levels of genes potentially regulated by the lncRNAs in ME/CFS

In order to elucidate potential mechanisms of actions of lncRNAs NTT, MIAT and EMX2OS in ME/CFS, we analyzed the expression profile of their potential downstream genes in patients and controls. NTT has been suggested to act on nearby genes, including IFNGR1 and PBOV1 [12]. ZEB1 has been reported to be downstream of MIAT; and EMX2 has been proposed to be regulated by EMX2OS [21, 27]. While EMX2 could not be detected in PBMCs (data not shown), ZEB1 level was found to have a mild but significant elevation in ME/CFS PBMCs as compared with controls (ME/CFS median ΔCT vs. control median ΔCT:7.60 vs. 7.82, p < 0.05, Fig. 4a). The expression levels of IFNGR1 and PBOV1 did not show significant differences between ME/CFS and controls (Fig. 4b, c). As for the correlation between the expressions of lncRNAs and downstream genes, a positive relationship between ZEB1 and MIAT level was detected using linear regression analysis in ME/CFS (r2= 0.47, p < 0.0001), but not in controls (Fig. 5a, b). Similarly, IFNGR1 level was found to be positively correlated with NTT level in ME/CFS (r2= 0.46, p < 0.0001), but not in controls (Fig. 5c, d). The linear regression analysis between NTT and PBOV1 showed no statistical significance both in ME/CFS and controls (Fig. 5e, f).

Fig. 4 Expression level of genes potentially regulated by MIAT (a) or NTT (b, c) in PBMCs of ME/CFS patients and controls. *p < 0.05 by Mann–Whitney U test, lines represent medians Full size image

Fig. 5 Correlation between lncRNA and downstream gene expressions. Correlation between MIAT and ZEB1 levels in ME/CFS (a) and in controls (b). Correlation between NTT and IFNGR1 in ME/CFS (c) and in controls (d). Correlation between NTT and PBOV1 levels in ME/CFS (e) and in controls (f) Full size image

Upregulation of NTT and MIAT by H 2 O 2 and poly (I:C) in human monocytic and glioma cell lines

Oxidative stress and recurrent herpes viral infection are known to contribute to ME/CFS pathology [28]. To investigate the potential effect of these stressors on the expression of NTT, MIAT and EMX2OS, we used human monocytic cell line THP-1 and glioma cell line KALS1 as models, and treated them with titrations of H 2 O 2 (oxidative stress) or poly (I:C) (TLR3 agonist, mimicking herpes viral infection) for 6 h. While NTT and MIAT were upregulated after stimulations, EMX2OS could not be detected in both cell lines. In THP-1 cells, NTT expression level increased to a mean of 1.47- and 3.06-fold after stimulation with 10 nM H 2 O 2 and 100 μM poly (I:C), respectively (Fig. 6a). Furthermore, MIAT level increased to a mean of 1.26- and 3.38-fold after THP-1 stimulation with 10 nM H 2 O 2 and 100 μM poly (I:C), respectively (Fig. 6b). Higher expression levels of the potential NTT downstream gene PBOV1 and the potential MIAT downstream gene ZEB1 were also detected in THP-1 after stimulations with 10 nM H 2 O 2 and 100 μM poly (I:C), respectively; however, the level of IFNGR1 (another potential NTT downstream gene) did not show an obvious change after both stimulations (Fig. 6c–e). In KALS1 cells, NTT could be upregulated to a mean of 1.49-fold when H 2 O 2 was titrated to 100 μM, and NTT level also increased to a mean of 1.65-fold after 50 μM poly (I:C) treatment (Fig. 6f). As for MIAT expression in KALS1, a 1.26-fold and a 1.31-fold increase were observed after 6 h 10 nM H 2 O 2 and 100 μM poly (I:C) stimulations, respectively (Fig. 6g). The expression levels of potential NTT and MIAT downstream genes after H 2 O 2 and poly (I:C) stimulations in KALS1 showed a similar pattern as those observed in THP-1 (Fig. 6h–j).