Expression of miR-146a and miR-155 in the urinary sediment of systemic lupus erythematosus
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- Wang, G., Tam, L., Kwan, B.C. et al. Clin Rheumatol (2012) 31: 435. doi:10.1007/s10067-011-1857-4
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We studied the levels of miR-146a and miR-155 in the urine sediment of SLE patients. The levels of miR-146a and miR-155 in the urine sediment of 40 SLE patients who were receiving calcitriol treatment and 13 healthy controls were determined with real-time quantitative polymerase chain reaction. The levels of urinary miR-146a and miR-155 in patients with SLE were significantly higher than that in healthy controls. Calcitriol treatment reduced the levels of urinary miR-155 in patients with SLE. The level of urinary miR-146a significantly correlated with estimated glomerular filtration rate (r = 0.242, P = 0.008). The level of urinary miR-155 significantly correlated with proteinuria (r = 0.407, P < 0.001) and systemic lupus erythematosus disease activity index (r = 0.278, P = 0.002). The level of urinary miR-146a reversely correlated with the urinary expression of TNF-α (r = −0.247, P = 0.012). Our results suggested that miR-146a and miR-155 might play important roles in the pathophysiology of SLE and the levels of urinary miR-146a and miR-155 could be used as potential markers for diagnosis, disease activity, and therapeutic response.
Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease characterized by disorder of the generation of auto-antibodies to components of the cell nucleus [1–3]. Although genetic, racial, hormonal, and environmental factors contribute to the development of SLE, the exact etiology of this devastating condition is unknown .
Recent studies showed that microRNAs (miRNAs), a group of small non-coding, single-stranded RNA molecules that regulate gene expression at post-transcriptional level by degrading or blocking translation of messenger RNA (mRNA) , play important roles in the pathogenesis of various autoimmune diseases [6–8]. In two studies, Dai et al. [9, 10] found that a number of miRNA species were differentially regulated in the peripheral blood mononuclear cells (PBMCs) and renal tissue of SLE patients. Amongst the known miRNA species, miR-146a and miR-155 are reported to be important regulators of the immune system [6–8]. Notably, Tang et al.  reported that the level of miR-146a in PBMC significantly correlated with disease activity in SLE patients, and manipulation of miR-146a might provide useful therapeutic interventions for SLE. In the present study, we explore the role of miR-146a and miR-155 in urine sediment as biomarkers of SLE.
Patients and methods
We studied 40 SLE patients between June 2008 and October 2008 in the Prince of Wales Hospital, Hong Kong. The study was approved by the Clinical Research Ethical Committee of the Chinese University of Hong Kong and all patients provided informed consent. All the patients were diagnosed according to the American College of Rheumatology diagnostic criteria and required maintenance immunosuppressive therapy. All the patients were treated with calcitriol at 0.25 μg per day for the prevention of osteoporosis. A whole-stream early morning urine specimen was collected for miRNA and mRNA expression study during clinic follow-up. All patients were followed for 6 months. Clinical data including serum creatinine, urea, 24-h urine protein, and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) were recorded at 0 and 3rd and 6th month. Glomerular filtration rate (GFR) was estimated by a standard equation . Treatment for individual patient was determined by a responsible physician and not affected by this study. All physicians were blinded from the results of RNA expression. We also studied the urine from 13 healthy subjects (all allied staff of our hospitals) as controls.
Urine specimens were collected and sent to laboratory for processing immediately or stored at 4°C overnight. Urine samples were then centrifuged at 3,000 g for 30 min and at 13,000 g for 5 min at 4°C. Supernatant was discarded and the urinary cell pellets were lysed by RNA lysis buffer (Qiagen Inc, Ontario, Canada). Specimens were then stored at −80°C until use.
Measurement of miRNA and mRNA levels
MirVana™ miRNA isolation kits (Ambion, Inc. Austin, TX, USA) were used for the extraction of total RNA from urinary sediment according to the manufacturer’s protocol. We confirmed the quantity and purity of urinary RNA by the value of optical density (OD) at 260 and 280 nm using a spectrometer (Hitachi, Japan).
TaqMan® miRNA reverse transcription Kits (Applied Biosystems, Foster City, CA, USA) and High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA) were used for reverse transcription. For miRNA, 1.67 μl total RNA was mixed with 1 μl specific primers, 0.05 μl 100 mM dNTPs (with dTTP), 0.5 μl 10× reverse transcription buffer, 0.33 μl (50 U) MultiScribe™ Reverse Transcriptase, 0.06 μl RNase inhibitor (20 U/μl) and made up to 5 μl with H2O. Reverse transcription was performed at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. For mRNA, 10 μl total RNA was mixed with 2 μl specific primers, 0.8 μl 100 mM dNTPs (with dTTP), 2 μl 10× reverse transcription buffer, 1 μl (50 U) MultiScribe™ Reverse Transcriptase, 1 μl RNase inhibitor (20 U/μl) and made up to 20 μl with H2O. Reverse transcription was performed at 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min. The resulting cDNA was stored in −80°C until use.
Urinary expression of miR-146a and miR-155, together with mRNA of inteleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were quantified by real-time quantitative polymerase chain reaction (RT-QPCR) using the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Commercially available Taqman primers and probes, including two unlabeled PCR primers and one FAM™ dye-labeled TaqMan® MGB probe were used for all the targets (all from Applied Biosystems). For mRNA expression, the primer and probe set was deliberately designed across the intron–exon boundary so as not to detect probable genomic DNA. For RT-QPCR, 2.5 μl universal master mix, 0.25 μl primer and probe set, 0.33 μl cDNA, and 1.92 μl H2O were mixed to make a 5-μl reaction volume. Each sample was run in triplicate. RT-QPCR were performed at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. β-Glucuronidase (GUSB, Applied Biosystems) and RNU48 (Applied Biosystems) were used as house-keeping genes to normalize the messenger RNA and microRNA expression, respectively. Results were analyzed with Sequence Detection Software version 2.3 (Applied Biosystems). In order to calculate the differences of expression level for each target among samples, the ΔΔCT method for relative quantitation was used. The average expression level of normal subjects was used as calibrator for urinary expression and the expression level of targets was a ratio relative to that of the controls.
Statistical analysis was performed by SPSS for Windows software version 13.0 (SPSS Inc., Chicago, IL, USA). All the results were presented in mean ± SD for data normally distributed and median (lower and upper quartiles) for the others. Since data of gene expression levels were highly skewed, either log transformation or nonparametric statistical methods were used. We used Mann–Whitney U test to compare the gene expression levels between groups and Spearman’s rank-order correlations to test associations between gene expression levels and clinical parameters. When no detectable level of a transcript was found (defined as no detectable level after 40 cycles of RT-QPCR) and there was zero value, a value equal to half of the minimum observed gene expression level was assigned. A P value of below 0.05 was considered as statistically significant. All probabilities were two-tailed.
Summary of clinical data
0.38 ± 0.77
0.37 ± 0.79
0.27 ± 0.45
Serum creatinine (μmol/l)
82.1 ± 37.1
92.8 ± 49.3*
94.3 ± 68.4*
GFR (ml/min/1.73 m2)
81.5 ± 27.9
74.2 ± 28.4*
77.6 ± 30.0*
0.88 ± 0.23
0.90 ± 0.23
0.90 ± 0.28
0.20 ± 0.10
0.22 ± 0.14
0.21 ± 0.12
1.68 ± 2.90
2.28 ± 2.92
1.95 ± 2.09
No. of cases
48.4 ± 12.6
31.8 ± 4.0
Urinary sediment expression of miRNAs
Correlations with clinical parameters
Correlations with urinary cytokine gene expression
Correlations between the levels of urinary miRNA and cytokine gene expression
r = −0.102, P = 0.304
r = −0.110, P = 0.268
r = −0.057, P = 0.566
r = −0.077, P = 0.441
r = −0.247, P = 0.012
r = −0.057 P = 0.564
Change in urinary miRNA in response to calcitriol
Previous studies have proved that miR-146a and miR-155 are expressed in many immune cell types such as B, T cells, monocytes, macrophages, and dendritic cells and are two important regulators in both innate and adaptive immunity including the development and differentiation of immnue cells, antibody production, and the inflammatory mediator release [13, 14]. Therefore, dysregulation of these miRNAs might participate in the pathogenesis of autoimmune disease in humans. Indeed a number of recent studies have discovered increased miR-146a and miR-155 expression in both peripheral blood mononuclear cells and synovial fibroblast of rheumatoid arthritis (RA) patients as compared with controls [15, 16]. In line with these previous findings, we further revealed that the levels of miR-146a and miR-155 in the urine sediment of SLE patients were higher than those of normal controls in the present study. On the contrary, another study demonstrated a decreased level of miR-146a in SLE patients, as compared with healthy controls . These contradictory results might reflect a difference in the overall miRNA profiles between urinary cells and PBMCs or different cytokine profiles between RA and SLE because cytokines are inducible factors for the cellular expression of these miRNAs . Nonetheless, these results showed that abnormal expression of miR-146a and miR-155 might be involved in the pathophysiology of SLE and levels of miR-146a and miR-155 in the urine sediment might be used as diagnostic markers for SLE. Besides, the strong correlations between the expression of urinary miR-146a and miR-155 suggested that these miRNAs might act synergistically in the regulation of immune response in SLE because both miRNAs play important roles in the regulation of immune response; however, further experiments are needed to verify this notion.
We also found that the expression of urinary miR-146a significantly correlated with estimated GFR and miR-155 significantly correlated with proteinuria and SLEDAI. These results support the previous hypothesis that these miRNAs are mostly induced in more active disease and may function as components of negative feedback loops in immune response and limit excessive inflammation [18, 19]. In fact, we found that there was a significantly inverse correlation between the levels of urinary miR-146a and TNF-α. In addition, these correlations indicated that levels of urinary miR-146a and miR-155 might be used as potential noninvasive markers of SLE disease activity. Studies involving a larger patient cohort and patients with more active SLE are needed for further confirmation.
In the present study, we found that the levels of urinary miR-155 decreased after calcitriol treatment. The underlying mechanism of this observation is uncertain. However, 1,25-dihydroxycholecalciferol (1, 25-(OH)2D3) has been proved to exert exquisite immunoregulatory properties besides its central roles in calcium and bone metabolism . A recent study further proved the altered expression of both miR-146a and miR-155 in dendritic cells after calcitriol treatment . These findings suggested that the possible immunomodulatory effects exerted by calcitriol in patients with SLE might partly attribute to its ability to influence the expression of miR-146a and miR-155 in certain immune cells and the levels of urinary miR-146a and miR-155 might be used to monitor treatment response.
There are a few limitations of our study. First, we detected the expression levels of the studied miRNAs using urine sediment without determining the cellular sources for each of them. Though both miRNAs have been frequently studied in cells related to immune response, they might express in other cell types [22, 23]. Future studies would be necessary to investigate the miRNA expression level in specific cell type in urine sediment. Second, we did not conduct a function study of these miRNAs. The underlying mechanism of the comparisons and correlations in this study needs further investigation.
In addition, we did not measure the serum levels of IL-1β, IL-6, or TNF-α and compared to their corresponding urinary expression. Previous studies from our group suggested that urinary cytokine gene expression does not correlate much with their serum level but does go in parallel with the intra-renal level of the corresponding cytokine and reflects the degree of intra-renal inflammation [24, 25]. Similarly, we did not measure the miR-146a and miR-155 expression in peripheral blood and compare those to the urinary expression. Our previous study, however, showed that the correlation between serum and urinary expression of other miRNA species is usually modest at best , but urinary expression may be a superior marker of the severity of renal involvement.
In summary, we found the urinary expression of miR-146a and miR-155 to be upregulated in patients with SLE. The expression of urinary miR-146a significantly correlated with the estimated GFR and miR-155 significantly correlated with proteinuria and SLEDAI. We also found the levels of urinary miR-155 to be decreased with calcitriol treatment. Our results suggested miR-146a and miR-155 might play an important role in the pathophysiology of SLE and the levels of urinary miR-146a and miR-155 could be potential markers for diagnosis, disease activity, and therapeutic response.
This study was supported in part by the National Natural Science Foundation of China (project 81000287) and CUHK research account 6901031.