Despite growing evidence that large intergenic noncoding RNAs (lincRNAs) can regulate gene expression and widely take part in normal physiological and disease conditions, our knowledge of systemic lupus erythematosus (SLE)-related lincRNAs remains limited. The aim of this study was to detect the levels of four lincRNAs (ENST00000500949: linc0949, ENST00000500597: linc0597, ENST00000501992: linc1992, and ENST00000523995: linc3995) involved in innate immunity in the peripheral blood mononuclear cells (PBMCs) of patients with SLE and correlate these lincRNA levels with disease activity, organ damage, clinical features and medical therapies.
Methods
PBMCs were obtained from 102 patients with SLE, 54 patients with rheumatoid arthritis (RA) and 76 healthy donors. lincRNA expression levels were measured by real-time quantitative polymerase chain reaction. Disease activity was assessed using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) scores, and organ damage was evaluated with the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index.
Results
linc0949 and linc0597 were significantly decreased in patients with SLE compared with patients with RA and healthy control subjects. linc0949 was correlated with SLEDAI-2K score (r = −0.329, P = 0.0007), as well as with complement component C3 level (r = 0.348, P = 0.0003). The level of linc0949 was also reduced in patients with SLE who had the presence of cumulative organ damage. In addition, decreasing expression of linc0949 was associated with lupus nephritis. linc0949 expression significantly increased after treatment, whereas neither disease activity nor organ damage correlated with linc0597 expression.
Conclusions
Our results provide novel empirical evidence that linc0949 could be a potential biomarker for diagnosis, disease activity and therapeutic response in SLE.
The online version of this article (doi:10.1186/s13075-015-0632-3) contains supplementary material, which is available to authorized users.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YFW, YJT and NS conceived of and designed the experiments and analyzed the data. YFW, FFZ, YJT and NS wrote the paper. YFW, FFZ, JYM, XYZ, LLW, BQ, SWX and SLC performed the experiments, collected blood samples and contributed reagents, materials and analytic tools. JYM, XYZ, LLW, BQ, SWX and SLC helped to draft the manuscript. All authors read and approved the final manuscript.
Abkürzungen
ACR
American College of Rheumatology
ANA
Antinuclear antibody
AZA
Azathioprine
C3
Complement component 3
CsA
Cyclosporine A
CYC
Cyclophosphamide
dsDNA
Double-stranded DNA
DXM
Dexamethasone
EDTA
Ethylenediaminetetraacetic acid
FBS
Fetal bovine serum
FK506
Tacrolimus
GAS5
Growth arrest–specific 5
hpf
High-power field
IFN
Interferon
IL
Interleukin
LEF
Leflunomide
linc0597
ENST00000500597
linc0949
ENST00000500949
linc1992
ENST00000501992
linc3995
ENST00000523995
lincRNA
Large intergenic noncoding RNA
LN
Lupus nephritis
lncRNA
Long noncoding RNA
miRNA
microRNA
MMF
Mycophenolate mofetil
MTX
Methotrexate
NS
Not significant
nt
Nucleotide
PBMC
Peripheral blood mononuclear cell
RA
Rheumatoid arthritis
RNP
Ribonucleoprotein
RPL13A
Ribosomal protein L13A gene
RT-qPCR
Real-time quantitative polymerase chain reaction
SDI
Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index
SEM
Standard error of the mean
SLE
Systemic lupus erythematosus
SLEDAI-2K
Systemic Lupus Erythematosus Disease Activity Index 2000
SLICC
Systemic Lupus International Collaborating Clinics
Sm
Smith
SSA
Sjögren’s syndrome–related antigen A
SSB
Sjögren’s syndrome–related antigen B
TLR
Toll-like receptor
TNF
Tumor necrosis factor
SEM
Standard error of the mean
Introduction
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with various clinical manifestations affecting different tissues. It is characterized by the deposition of immune complexes due to widespread loss of immune tolerance to nuclear self-antigens, as well as by excessive proinflammatory cytokine production and damage to multiple organ systems [1]. Recent experimental and clinical studies have placed new emphasis on the role of the innate immune system in SLE. It has become apparent that Toll-like receptors (TLRs) can participate in cell activation by self molecules such as immune complexes containing DNA or RNA. Indeed, TLRs have an important role in the pathogenesis of lupus involving recruitment of adapter proteins; activation of protein kinases and transcription factors; and expression of inflammatory cytokines, chemokines, endothelial adhesion molecules and costimulatory molecules [2]. TLR signaling also stimulates B cell proliferation, cell differentiation and immunoglobulin class switching [2,3].
In the past, the importance of non-protein-coding RNAs has been emphasized in many biological and pathological processes [4]. Much research has been focused on microRNAs (miRNAs). miRNAs are small RNA molecules with a length of approximately 22 nucleotides (nt) that play a critical role in the pathogenesis of SLE by regulating gene expression at posttranscriptional levels [5,6]. miRNAs have also been reported to be involved in the local inflammatory response that ultimately leads to tissue injury and organ damage [7]. Recently, several studies have shown the feasibility of using miRNAs as biomarkers in body fluids for the diagnosis of SLE [8-10]. Though miRNAs play important roles in SLE, they are only a small fraction of the noncoding regions of the mammalian genome. Unlike miRNAs, long noncoding RNAs (lncRNAs) are expressed abundantly, including large intergenic noncoding RNAs (lincRNAs) [11].
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lncRNAs are a class of mRNA-like transcripts ranging in length from 200 nt to over 100 kb and lacking any significant open reading frames [12,13]. They are highly diverse and actively present in virtually every aspect of cell biology, such as cell differentiation, cell proliferation, DNA damage response, dosage compensation and chromosomal imprinting. Recently, a number of lncRNA molecules have been reported to be involved in diverse diseases [14-16]. Some evidence indicates that a few samples of lncRNAs could regulate the immune system [17-19]. In particular, there are several emerging hypotheses on lncRNA involvement in rheumatic diseases, such as rheumatoid arthritis (RA) [20,21], autoimmune thyroid disease [22] and psoriasis [23]. Other preliminary data in a murine model system pointed to a link between the lncRNA growth arrest–specific 5 (GAS5) and disease susceptibility to SLE [16]. In addition, the chromosomal locus of GAS5, 1q25, was showed to be associated with human SLE development in genetic studies [23-25].
Because of the heterogeneous presentation of patients with SLE and their unpredictable disease course, there is a pressing need to identify biomarkers that will facilitate better diagnosis and prognosis, and lincRNAs as biomarkers are still largely unexplored in this regard. It has been reported that four lincRNAs (linc0949, linc0597, linc1992 and linc3995) not only are differentially expressed following innate activation of THP-1 macrophages but also regulate induction of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 [26]. Moreover, it is well-established that IL-6 and TNF-α are involved in SLE pathogenesis [27-29].
As mentioned above, we hypothesized that these lincRNAs would produce cross-linking with SLE via innate immunity and play a critical role in the pathogenesis of SLE and that they might serve as biomarkers of disease activity, organ damage and medical response. In the present study, we aimed to investigate whether the expression levels of these lincRNAs in peripheral blood mononuclear cells (PBMCs) were abnormal in patients with SLE, assess the relationship of the levels with disease activity and organ damage, and explore new biomarkers used in disease monitoring and prognostication.
Methods
Patients and healthy controls
All samples from patients with SLE and patients with RA were obtained from the Department of Rheumatology of Renji Hospital (Shanghai, China). All patients with SLE met at least four of the American College of Rheumatology (ACR) 1982 revised criteria for SLE [30]. Patients with RA were diagnosed according to the ACR/European League Against Rheumatism 2010 classification criteria for RA [31]. The control group comprised healthy volunteers with no history of autoimmune disease or immunosuppressive therapy. Otherwise eligible individuals with a current or recent infection were excluded from the study. Control subjects were frequency-matched with the patients for age and sex. All participants were from the Han Chinese population. The study was approved by the Research Ethics Board of Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Informed consent was obtained from all study participants.
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The patients with lupus were all receiving steroid therapy at the time of the study, and a prednisone dosage per day (dosages of other steroids were converted to prednisone equivalents) from 2.5 mg to 500 mg (mean dosage: 29.5 mg/day). In addition, 42 patients were receiving immunosuppressive therapy (azathioprine (AZA; n = 7), cyclophosphamide (CYC; n = 10), cyclosporine A (CsA; n = 6), tacrolimus (FK506; n = 1), leflunomide (LEF; n = 2), mycophenolate mofetil (MMF; n = 8), methotrexate (MTX; n = 8)), and 59 were receiving an antimalarial drug (chloroquine or hydrochloroquine). For each patient, the severity of disease was assessed with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) [32]. Organ damage (defined as nonreversible change, not related to active inflammation, occurring since the onset of lupus and present for at least 6 months) was assessed using the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI) score [33]. In our cohort, nearly 51.0% of patients (52 of 102 patients with SLE) had either previous or current lupus nephritis (LN) (Table 1). Subjects were considered to have active renal disease if proteinuria was ≥0.5 g/day, hematuria was ≥5 red blood cells per high-power field (hpf), pyuria was ≥5 white blood cells/hpf or cellular casts were present. Infection, kidney stones and other causes were excluded.
Table 1
Large intergenic noncoding RNAlinc0949by presence or absence of clinical features of systemic lupus erythematosusa
Clinical features
SLE clinical features present
SLE clinical features absent
P-value
N
Mean ± SEM (range, 10−3)
N
Mean ± SEM (range, 10−3)
Renal
50
2.16 ± 0.152
52
1.47 ± 0.132
0.0014
Rash
33
1.89 ± 0.200
69
1.87 ± 0.199
NS
Arthritis
35
1.79 ± 0.327
67
1.97 ± 0.163
NS
Serositis
21
1.81 ± 0.307
81
1.72 ± 0.364
NS
Mucosal ulcer
19
2.02 ± 0.207
83
1.87 ± 0.187
NS
Hematologic
30
1.84 ± 0.243
72
2.06 ± 0.179
NS
Neurologic
9
1.99 ± 0.084
93
2.05 ± 0.45
NS
Autoantibodies
Anti-dsDNA
40
1.91 ± 0.274
62
1.88 ± 0.264
NS
Anti-Sm
16
2.07 ± 0.202
86
1.78 ± 0.193
NS
Anti-nucleosome
40
1.91 ± 0.204
62
1.88 ± 0.212
NS
Anti-SSA/SSB
32
2.03 ± 0.258
70
2.01 ± 0.176
NS
Anti-RNP
22
1.95 ± 0.253
80
1.96 ± 0.165
NS
Medical therapy
Prednisone dose ≥30 mg/day
48
2.08 ± 0.175
54
1.94 ± 0.181
NS
Immunosuppressantsb
42
2.00 ± 0.141
60
1.54 ± 0.151
0.0365
adsDNA, Double-stranded DNA; NS, Not significant; RNP, Ribonucleoprotein; SEM, Standard error of the mean; SLE, Systemic lupus erythematosus; Sm, Smith; SSA, Sjögren’s syndrome–related antigen A; SSB, Sjögren’s syndrome–related antigen B. bImmunosuppressants included azathioprine, cyclophosphamide, cyclosporine A, FK506 (tacrolimus), leflunomide, mycophenolate mofetil and methotrexate.
Peripheral blood samples handling and RNA processing
Peripheral blood samples (10 ml) were obtained from each subject. The samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA). PBMCs were isolated from anticoagulated whole blood by use of Ficoll density gradient centrifugation. Then total RNA was extracted from PBMCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The integrity of the RNA was assessed using capillary gel electrophoresis, and the concentrations of RNA were measured using a NanoDrop™ 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) with a 260 nm/280 nm ratio above 1.8. About 200 ng of total RNA were reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara Bio, Dalian, China). All RNA and cDNA samples were stored at −70°C before use.
Cell culture and stimulation
Peripheral blood samples were obtained from five healthy donors and five patients with SLE. The samples were collected in tubes containing EDTA. PBMCs were isolated from anticoagulated whole blood by Ficoll density gradient centrifugation. Two hours before stimulation, 1 × 106 PBMCs were cultured in 24-well flat-bottomed plates in 500 μl of RPMI 1640 medium containing 10% fetal bovine serum (FBS). Then the PBMCs were stimulated for 4 hours with the TLR2 ligand Pam3CK4 (20 ng/ml).
Cell culture and treatment with dexamethasone and immunosuppressant agents
Peripheral blood samples were obtained from two healthy donors. PBMCs were isolated from anticoagulated whole blood by use of Ficoll density gradient centrifugation. PBMCs (1 × 106) were resuspended for 2 hours in 500 μl of RPMI 1640 medium containing 10% FBS, then dexamethasone was added with the indicated concentration (Dexamethasone concentrations were 10ng/ml,100ng/ml,1000ng/ml,10ug/ml, respectively) for another 24 hours, as were CsA (200 nmol) and FK506 (20 nmol). RNA samples were then isolated, and real-time quantitative PCRs (RT-qPCRs) were performed.
Real-time quantitative polymerase chain reaction
To quantify the expression of four lincRNAs (linc0949, linc0597, linc1992 and linc3995), cDNA was amplified by RT-PCR with SYBR Green (SYBR Premix Ex Taq RT-PCR kit; Takara Bio). The primer sequences used for SYBR Green–based RT-PCR are given in Table 2. The ribosomal protein L13A (RPL13A) gene was used as an internal control to normalize the amounts of cDNA. The SYBR Green assays were performed in duplicate using an ABI ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative expression levels were calculated using the 2−ΔCt comparative threshold cycle method.
Table 2
Primers used to amplify transcripts of large intergenic noncoding RNAsa
Gene
Forward
Reverse
RPL13A
5′-CTGGAGGAGAAGAGGAAAGAGA-3′
5′-TTGAGGACCTCTGTGTATTTGTCAA-3′
ENST00000500597
5′-TTGGATTCATCCCGTTCACCTCCA-3′
5′-CAGCATGACGATCAAGCGAGATTC-3′
ENST00000501992
5′-AACTCCTGACCTCAGGTGATCCAT-3′
5′-AAGGGAGTTTCAGAAGGTGTGGCT-3′
ENST00000500949
5′-TCCTGCAACCCAAGGTGGATACTT-3′
5′-CTGCAGTGAGCAGAAATCACGCAT-3′
ENST00000523995
5′-GTTTGTGGCATATGGCTCTGCTGT-3′
5′-CATTGCAGGAAAGAGTGCCAAGGT-3′
aThe four large intergenic noncoding RNAs primers are derived from the literature [26].
Statistical analysis
Data were analyzed with GraphPad Prism version 5.0 software (GraphPad Software, La Jolla, CA, USA). The nonparametric Mann–Whitney U test was used to compare gene expression between two groups. The correlation between groups was evaluated using Spearman’s rank correlation coefficient test. The strength of the correlation was graded using Cohen’s criteria as follows: 0.3 to 0.5 = weak, 0.5 to 0.7 = moderate and >0.7 = strong [34]. P-values (two-tailed) <0.05 were considered statistically significant.
Results
Decreased linc0949 and linc0597 levels in patients with systemic lupus erythematosus
The expression levels of four lincRNAs (linc0949, linc0597, linc1992 and linc3995) in PBMCs taken from 102 patients with SLE, 54 patients with RA and 76 healthy donors were measured using RT-qPCR. Patients with SLE and healthy donors did not differ significantly with respect to mean age or sex distribution (Table 3). The average disease duration of patients enrolled in our study was 4.98 years for those with SLE and 5.12 years for those with RA. In general, the patients with SLE had mild to moderate flares of disease activity and severity, with a mean SLEDAI-2K score of 7 and a mean SDI of 0.78 (Table 3).
Table 3
Demographic dataa
Patients with SLE (n = 102)
Patients with RA (n = 54)
Healthy donors (n = 76)
Age (yr)
34.3 ± 1.3 (16 to 65)
38.7 ± 2.3 (20 to 65)
34.0 ± 1.2 (20 to 63)
Sex (n)
Female
93
46
70
Male
9
8
6
Disease duration (yr)
4.98 ± 0.76 (0.04 to 28)
5.12 ± 0.84 (0.5 to 20)
–
ANA (%)
95.0
–
–
SLEDAI-2K
6.82 ± 0.52 (1 to 15)
–
–
SDI
0.78 ± 0.14 (0 to 3)
–
–
aANA, antinuclear antibody; RA, Rheumatoid arthritis; SDI, Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index; SLE, Systemic lupus erythematosus; SLEDAI-2K, Systemic Lupus Erythematosus Disease Activity Index 2000 score.
As shown in Figure 1A, patients with SLE had significantly lower linc0949 levels than healthy donors and patients with RA (both P < 0.0001). Also, the expression of linc0597 was decreased dramatically in patients with SLE compared with healthy donors and patients with RA (P = 0.0001 and P < 0.0001, respectively) (Figure 1B). Figure 1C and Figure 1D, however, show no significant differences in linc1992 and linc3995 levels between patients with SLE and healthy donors or patients with RA. These results revealed that lower expression of linc0949 and linc0597 was specific to SLE, so we selected these lincRNAs for further research.
Figure 1
Comparison of expression of four large intergenic noncoding RNAs (linc0949, linc0597, linc1992 and linc3995) between patients with SLE, patients with RA and healthy donors. Each symbol represents an individual patient; horizontal lines indicate median values. The expression levels of the four large intergenic noncoding RNAs (lincRNAs) in 102 patients with SLE, 54 patients with RA and 76 healthy donors were analyzed by real-time quantitative PCR and normalized by RPL13A level. (A) Decreased expression of linc0949 in patients with SLE versus patients with RA and healthy donors. (B)linc0597 expression was significantly reduced in patients with SLE versus patients with RA and healthy donors. No apparent difference was detected in the expression of linc1992(C) and linc3995(D) between patients with SLE, patients with RA and healthy donors. NS, Not significant. ***P < 0.001. HD, Healthy donors; RA, Rheumatoid arthritis; SLE, Systemic lupus erythematosus.
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Association of linc0949 level with disease activity in patients with systemic lupus erythematosus
To investigate whether the expression of linc0949 and linc0597 is related to SLE disease activity, we compared the relative expression levels of the lincRNAs in patients with SLE with different levels of disease activity, assessed on the basis of SLEDAI-2K score and the level of complement C3. In accordance with the SLEDAI-2K flare system, patients with SLE were divided into those with stable disease (SLEDAI-2K scores from 0 to 4), those with a mild flare (SLEDAI-2K scores from 5 to 10) and those with a moderate to severe disease flare (SLEDAI-2K scores >10). linc0949 was significantly lower in patients with SLE who had a mild flare or a moderate to severe flare of disease than in patients without a flare (P = 0.0032 and P = 0.0004, respectively) (Figure 2A). In addition, a correlation between linc0949 and SLEDAI-2K score was observed in that decreased levels of linc0949 coincided with increased SLEDAI-2K score in patients with SLE (r = −0.329, P = 0.0007) (Figure 2B).
Figure 2
Association of large intergenic noncoding RNA linc0949 expression with disease activity in patients with systemic lupus erythematosus. (A) Patients with systemic lupus erythematosus (SLE) with a moderate to severe flare of disease (Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score >10) or a mild flare of disease (SLEDAI-2K score from 5 to 10) had significantly lower large intergenic noncoding RNA linc0949 expression than did those without a disease flare (SLEDAI-2K score <4) at the time of blood donation. (B)linc0949 expression was negatively correlated with SLEDAI-2K score. (C)linc0949 expression was significantly decreased in patients with SLE with a reduced level of complement C3 (<80 mg/dl) compared with those with normal C3levels. (D) A significantly positive correlation was observed between linc0949 expression and C3 level in patients with SLE. **P < 0.01, ***P < 0.001.
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C3 level is also an indicator of disease activity. Hypocomplementemia is often observed in patients with SLE with active disease. In the present research, linc0949 expression was significantly decreased in patients with SLE who had a reduced level of complement C3 (<80 mg/dl) compared with those with normal levels of C3 (P < 0.0001) (Figure 2C). Further analysis revealed a positive correlation between linc0949 level and C3 level (r = 0.348, P = 0.0003) (Figure 2D). However, the level of linc0597 did not correlate with SLEDAI-2K and complement C3 level (data not shown). These results indicate that the abnormal expression of lincRNA0949 may be a key indicator of disease activity in patients with SLE.
Reduced expression of linc0949 in patients with systemic lupus erythematosus with organ damage
SLE is a chronic multisystem autoimmune disease that can affect virtually every organ system and may lead to significant morbidities. Assuming that lincRNAs are involved in tissue damage and inflammation, we investigated whether linc0949 and linc0597 were associated with different levels of chronic and irreversible organ damage in the patients with SLE. The results revealed significantly lower levels of linc0949 in patients with SLE with SDI scores of 1 to 2 and in those with scores >2 versus those without organ damage (P = 0.0059 and P = 0.0009, respectively) (Figure 3A). linc0597 expression was not significantly reduced in patients with organ damage (SDI ≥1) versus those who remained damage-free (data not shown).
Figure 3
Large intergenic noncoding RNA linc0949 expression was decreased in patients with systemic lupus erythematosus who had organ damage. (A) In our cohort, linc0949 was dramatically decreased in patients with systemic lupus erythematosus (SLE) who had chronic and irreversible organ damage (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI) scores 1 to 2 or more) compared with those with no damage. (B) Large intergenic noncoding RNA linc0949 expression exhibited a decreasing trend in patients with lupus nephritis (LN; n = 52) relative to patients with no history of LN (n = 50). (C) Patients with active LN had lower linc0949 expression than those without renal manifestations, whereas linc0949 level was not significantly different in patients with inactive LN compared with those without LN. NS, Not significant. **P < 0.01, ***P < 0.001.
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LN is one of the most common clinical manifestations and causes of organ damage in patients with SLE, so we wanted to know whether lincRNA levels are related to LN. In our cohort, nearly 51% of patients (52 of 102 patients with SLE) had either previous or current LN (Table 1). Patients with LN had lower linc0949 expression levels than those without renal manifestations (P = 0.0014) (Figure 3B). Expression levels of linc0949 were decreased in the group of patients with active LN compared with those without LN (P = 0.0009) (Figure 3C), whereas linc0949 levels were not significantly different in patients with inactive LN compared with patients without LN at the time of blood drawing (P = 0.0739) (Figure 3C). These data suggest that linc0949 expression is related with cumulative organ damage in SLE and that linc0949 may be useful in predicting long-term outcome and prognosis in patients with SLE.
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Relationship of large intergenic noncoding RNA levels with clinical manifestations and medical therapies
To assess the association between lincRNA levels and clinical manifestations, autoantibody profiles and medical treatments, linc0949 and linc0597 levels were compared between patients with certain clinical features and those without certain clinical features. We identified no association between linc0949 or linc0597 expression and clinical manifestations such as rash, arthritis, serositis, mucosal ulcer, hematologic involvement or neurologic manifestations (linc0949: Table 1; linc0597: data not shown). We also found that neither linc0949 nor linc0597 appeared to be associated with autoantibody production, including anti-double-stranded DNA, anti-Smith antibodies, antinucleosome antibodies, anti–Sjögren’s syndrome–related antigen A and B antibodies and antiribonucleoprotein antibodies (linc0949: Table 1; linc0597: data not shown).
When medical therapies were considered, the expression of linc0949 exhibited no significant difference in patients receiving medium to high doses of prednisone (>30 mg/day) compared with patients treated with low doses of prednisone (Figure 4A). By contrast, the expression of linc0949 in patients with SLE being treated with immunosuppressants (AZA, CYC, CsA, LEF, MMF, MTX and FK506) was significantly reduced compared with those not receiving immunosuppressants at the time of blood donation (P = 0.0365) (Figure 4B).
Figure 4
Association of large intergenic noncoding RNA linc0949 level with medical therapies in patients with systemic lupus erythematosus. (A) The level of the large intergenic noncoding RNA linc0949 had no apparent difference in patients with systemic lupus erythematosus (SLE) whose daily dosage of prednisone was >30 mg compared with those whose daily dosage of prednisone was <30 mg. (B)linc0949 expression decreased significantly in patients with SLE who had used immunosuppressants (azathioprine, cyclophosphamide, cyclosporine A (CsA), leflunomide, mycophenolate mofetil, methotrexate, FK506) compared with those without immunosuppressant treatment. (C) The expression of linc0949 was not related to the use of drugs. The data are from two healthy donors. (D)linc0949 expression was detected in three patients with active SLE at the beginning of and after 12 weeks of treatment. As we observed, the linc0949 expression of all three patients was notably increased when they achieved significant clinical improvement after treatment. *P < 0.05. DXM, Dexamethasone; NS, Not significant; Pred, Prednisone.
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We next sought to assess whether SLE disease activity or antirheumatic drugs affect the expression of linc0949, as it was found that some lncRNAs can be induced in response to the anti-inflammatory agent dexamethasone [35]. Thus, we treated PBMCs of two healthy donors with different concentrations of dexamethasone, CsA and FK506. FoxP3 level was significantly reduced after treatment (Additional file 1: Figure S1), which supported the fact that dexamethasone and immunosuppressive agents worked effectively in vitro. As shown in Figure 4C, dexamethasone, CsA and FK506 did not affect the expression of linc0949. This result relates to the effects of antirheumatic drugs on the expression of linc949, which confirms that linc0949 is intrinsically underexpressed in patients with SLE.
We next investigated whether linc0949 was responsive to treatment and changes over time in conjunction with disease activity. We chose three patients with SLE(P1,P2,P3), as described in Figure 4D, P1 and P3 had initial onset of biopsy-proved type IV LN, P2 had neuropsychological lupus, and their peripheral blood samples were collected at both the beginning of treatment and after 12 weeks of treatment. P1 and P3 used high-dose prednisone (1 mg/kg per day) plus mycophenolate mofetil (1.5-2 g/day), whereas P2 used repeated pulses of glucocorticoid (500 mg intravenous methylprednisolone per day) for three days and then prednisone (1 mg/kg per day) plus monthly pulse of cyclophosphamide (0.8 g/month). After treatment, all of the three patients achieved clinical remission, with the urinary protein level dropping to less than 0.5 g/24 hour in P1 and P3; and with cerebrospinal fluid inspection and head MRI restoring to normal in P2. SLEDAI-2K score of the three patients reduced to a stable level (Additional file 1: Figure S1). In concordance with the clinical improvement, linc0949 expression in these three patients also significantly increased (Figure 4D).
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Abnormal regulation of lincRNAs during innate activation of peripheral blood mononuclear cells in patients with systemic lupus erythematosus
To investigate the different response to innate immunity of linc0949 and linc0597 in healthy donors and patients with SLE, we stimulated PBMCs from healthy donors and patients with SLE with TLR2 ligands. We chose TLR2 ligands because it was reported that linc0949 and linc0597 were regulated in THP-1 macrophages following Pam3CSK4 stimulation [26] and TLR2 was required for the production of prototypical lupus autoantibodies and the development of renal disease in murine lupus [36,37]. To prepare cell samples, PBMCs obtained from 5 healthy donors and 5 patients with SLE were stimulated with Pam3CSK4 for 4 hours. Then we performed RT-qPCR to identify changes in lincRNA expression. In PBMCs of healthy controls, linc0949 was suppressed (Figure 5A), while linc0597 was increased (Figure 5B) after treatment with Pam3CSK4. But in patients with SLE, linc0949 and linc0597 could not response to the stimuli compared with healthy donors (Figure 5A and B). These results demonstrated that lincRNAs were indeed involved in the complex regulatory network of innate immunity.
Figure 5
Regulation of large intergenic noncoding RNAs in peripheral blood mononuclear cells by Pam3CSK4. (A) Expression of the large intergenic noncoding RNA linc0949 was lower in untreated and stimulated peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) than in those of healthy controls. linc0949 was suppressed by the Toll-like receptor 2 (TLR2) ligand Pam3CK4 (20 ng/ml) in healthy controls, and it was not significantly different after treatment in patients with SLE. (B)linc0597 was upregulated by TLR2 agonist Pam3CK4 (20 ng/ml) in PBMCs of healthy controls, whereas it did not respond to the stimuli in patients with SLE. linc0597 expression was significantly decreased in PBMCs of patients with SLE compared with healthy controls both before stimulation and after treatment. The data shown are from five healthy donors and five patients with SLE after incubation with the indicated stimuli for 4 hours. Real-time quantitative PCR of the indicated RNAs is normalized to RPL13A levels (mean ± standard deviation (SD)). Data are mean ± SD. P-values were determined by performing unpaired t-tests. NS, Not significant. *P < 0.05; **P < 0.01.
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Discussion
In recent years, an increasing body of evidence has shown that lncRNAs play major biological roles in embryogenesis, stem cell biology and cellular development and show developmental and tissue-specific expression patterns [11,38,39]. Studies have also suggested that abnormal expression of lncRNAs might be associated with numerous diseases, indicating that these RNAs may open a new avenue for diagnostic and therapeutic targets by recognition of their roles in human disease.
In the present study, we detected four lincRNAs (linc0949, linc0597, linc1992 and linc3995) and investigated the association between their expression levels and specific clinical features of SLE. Two of these lincRNAs (linc0949 and linc0597) were significantly decreased in patients with SLE compared with healthy donors and disease controls. linc0949 was associated with disease activity, as assessed using the SLEDAI-2K score and C3 level in patients with SLE. Moreover, linc0949 expression was reduced in patients with SLE with ongoing or cumulative organ damage, as assessed based on SDI score or the presence of active LN. linc0949 expression does not participate in clinical manifestations other than LN, which demonstrates that it has very good detection specificity for LN. Lower levels of linc0949 may thus be helpful to identify patients with SLE who have active and severe disease. To evaluate the effect of antirheumatic drugs on the expression of linc949, we used in vitro studies to test whether the addition of dexamethasone, CsA or FK506 to cultured PBMCs would affect the expression of linc0949. As shown in Figure 4C, these antirheumatic drugs did not affect the expression of linc0949 in PBMCs, which confirmed that linc0949 was intrinsically underexpressed in patients with SLE. linc0949 expression of three patients with severe disease flares significantly increased after treatment (Figure 4D), indicating that linc0949 might be responsive to treatment and might change in conjunction with disease activity and severity and suggesting that linc0949 might be used to monitor disease progression and guide therapy.
Over the past several decades, tremendous enthusiasm and efforts have been devoted to biomarkers for SLE because the diagnosis of SLE requires a combination of clinical manifestations and biomarkers and no single test is sufficiently sensitive and specific to be diagnostic. The traditional antibodies fail to identify the pathogenic processes, organ damage and biological responses to a therapeutic intervention. Many groups, including the members of our laboratory, have found a set of potential biomarkers for SLE. For example, interferon (IFN)-induced genes and IFN-inducible chemokines may serve as new biomarkers for active and severe disease in patients with SLE [40,41]. Some limitations of these biomarkers are revealed gradually, however. Several studies have shown that overexpressed transcripts of the type I IFN pathway are also identified in patients with myositis, RA, Sjögren’s syndrome and scleroderma [42-44], so an IFN signature or chemokine is not sufficiently specific. In two longitudinal studies to date, researchers have reported conflicting results on the correlations between type I IFN gene signature score and diseases activity [45,46]. There is an urgent need for SLE biomarkers that can help enhance comprehension of the mechanisms of diseases or effects of therapies by relating the changes of molecular and cellular pathways to disease status or clinical responses. In our present study, we demonstrate that lower expression of linc0949 is specific for SLE and that it is helpful in identifying disease activity, monitoring disease progression and guiding therapy. However, linc0949 needs to be further investigated in large-scale multicenter trials.
On the basis of our present observations, we believe that linc0949 could be a potentially readily accessible biomarker useful for diagnosing SLE. As a novel biomarker, lincRNAs have the following characteristics. First, lincRNAs display a wide range of stabilities in the samples comparable to those of mRNAs of protein-coding genes [47]. Second, they show a greater tissue specificity compared with protein-coding mRNAs and miRNAs, which are frequently expressed in multiple tissues, and they show highly increased or decreased expression levels in disease [15]. In addition, lincRNAs are also detectible in body fluids such as plasma and urine [48-50], diagnostic samples of which are easy to collect using noninvasive methods. Moreover, detection of the lincRNAs is simple, inexpensive and has high throughput, making it a suitable approach to gaining an overview of disease activity and severity in patients with SLE. These features make lincRNAs very suitable as biomarkers, and many studies have been published on this matter in recent years, both in cancer and in other human diseases such as cardiovascular diseases [50] and neurological disorders [51].
Most lncRNAs described to date have been found to be related to transcriptional regulation or mRNA processing, characteristics that they share with microRNAs. However, unlike microRNAs, lncRNAs show a greater complexity of their functions and have a wider spectrum of biological contexts, such as epigenetic regulation, enhancer-like function and RNA splicing, editing and export [52]. In our ongoing experiments, we found that linc0949 and linc0597 could be induced by TLR2 in PBMCs of healthy donors, but they did not respond to the stimuli in patients with SLE as compared with healthy donors (Figure 5). These results validate that lincRNAs were indeed involved in the complex regulatory network of innate immunity. We hypothesized that the regulation defect of linc0949 and linc0597 could contribute to the pathogenesis of SLE and that lincRNAs may provide potential novel strategies for therapeutic intervention, although their function and mechanism of action need further exploration.
We have suggested the abnormal expression of linc0949 in patients with SLE, as well as the association of lincRNA level with disease activity and organ damage; however, in this study, we did not conduct a functional study of this lincRNA, and the underlying mechanism needs further investigation. We did not detect the expression of linc0949 released in the local target tissues and in specific cell subsets in PBMCs. Future studies are needed to investigate the lincRNA expression level in specific organ and cell types as well.
Conclusions
We found that the expression of two lincRNAs was dramatically reduced in patients with SLE and that the decreasing level of linc0949 was correlated with disease activity, degree of organ damage and medical therapies in patients with SLE. linc0949 may serve as a potential biomarker for diagnosis, disease activity and therapeutic interventions in patients with SLE.
Acknowledgments
This work was supported by the National Basic Research Program of China (973 Program) (2014CB541901 and 2014CB541902), the National Natural Science Foundation of China (81025016, 81230072, 31370880 and No. 81421001), the State Key Laboratory of Oncogenes and Related Genes (91-14-05), the Key Research Program of the Chinese Academy of Sciences (KJZD-EW-L01-3) and the Program of the Shanghai Commission of Science and Technology (12ZR1435900, 12JC1406000 and 12431900703). The funders had no role in study design, data collection and analysis, the decision to publish or the preparation of the manuscript. The authors extend special thanks to Yuting Qin and Ting La of the Chinese Academy of Sciences for providing technological guidance during the course of this study.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YFW, YJT and NS conceived of and designed the experiments and analyzed the data. YFW, FFZ, YJT and NS wrote the paper. YFW, FFZ, JYM, XYZ, LLW, BQ, SWX and SLC performed the experiments, collected blood samples and contributed reagents, materials and analytic tools. JYM, XYZ, LLW, BQ, SWX and SLC helped to draft the manuscript. All authors read and approved the final manuscript.
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