B Cell-Related Circulating MicroRNAs With the Potential Value of Biomarkers in the Differential Diagnosis, and Distinguishment Between the Disease Activity and Lupus Nephritis for Systemic Lupus Erythematosus

Our understanding of circulating microRNAs (miRNAs) related to systemic lupus erythematosus (SLE) remains very limited. In this study, we screened SLE-specific miRNAs in plasma from 42 B cell-related miRNAs by using miRNA PCR Array. The selected miRNAs were first confirmed in plasma samples from 50 SLE patients, 16 rheumatoid arthritis (RA) patients, and 20 healthy donors using qRT-PCR. We then investigated the relationship between expressions of the selected miRNAs and SLE clinical indicators. As a result, 14 miRNAs (miR-103, miR-150, miR-20a, miR-223, miR-27a, miR-15b, miR-16, miR-181a, miR-19b, miR-22, miR-23a, miR-25, miR-92a, and miR-93) were significantly decreased in the plasma of SLE patients compared with healthy controls (P < 0.05) and could act as the diagnostic signature to distinguish SLE patients from healthy donors. Six miRNAs (miR-92a, miR-27a, miR-19b, miR-23a, miR-223, and miR-16) expressed in plasma were significantly lower in SLE patients than in RA patients (P < 0.05), revealing the potentially diagnostic signature to distinguish SLE patients from RA patients. Furthermore, the downregulated expression of miR-19b, miR-25, miR-93, and miR-15b was associated with SLE disease activity (P < 0.05) while miR-15b and miR-22 expressions were significantly lower in SLE patients with low estimate glomerular filtration rate (eGFR < 60 ml/min/1.73 m2) (P < 0.05). The diagnostic potential of miR-15b for SLE disease activity and lupus nephritis (LN) with low eGFR was validated on an independent validation set with 69 SLE patients and a cross-validation set with 80 SLE patients. In summary, the signature of circulating miRNAs will provide novel biomarkers for the diagnosis of SLE and evaluation of disease activity and LN.

Our understanding of circulating microRNAs (miRNAs) related to systemic lupus erythematosus (SLE) remains very limited. In this study, we screened SLE-specific miRNAs in plasma from 42 B cell-related miRNAs by using miRNA PCR Array. The selected miRNAs were first confirmed in plasma samples from 50 SLE patients, 16 rheumatoid arthritis (RA) patients, and 20 healthy donors using qRT-PCR. We then investigated the relationship between expressions of the selected miRNAs and SLE clinical indicators. As a result, 14 miRNAs (miR-103, miR-150, miR-20a, miR-223, miR-27a, miR-15b, miR-16, miR-181a, miR-19b, miR-22, miR-23a, miR-25, miR-92a, and miR-93) were significantly decreased in the plasma of SLE patients compared with healthy controls (P < 0.05) and could act as the diagnostic signature to distinguish SLE patients from healthy donors. Six miRNAs (miR-92a, miR-27a, miR-19b, miR-23a, miR-223, and miR-16) expressed in plasma were significantly lower in SLE patients than in RA patients (P < 0.05), revealing the potentially diagnostic signature to distinguish SLE patients from RA patients. Furthermore, the downregulated expression of miR-19b, miR-25, miR-93, and miR-15b was associated with SLE disease activity (P < 0.05) while miR-15b and miR-22 expressions were significantly lower in SLE patients with low estimate glomerular filtration rate (eGFR < 60 ml/min/1.73 m 2 ) (P < 0.05). The diagnostic potential of miR-15b for SLE disease activity and lupus nephritis (LN) with low eGFR was validated on an independent validation set with 69 SLE patients and a cross-validation set with 80 SLE patients. In summary, the signature of circulating miRNAs will provide novel biomarkers for the diagnosis of SLE and evaluation of disease activity and LN. inTrODUcTiOn Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease which affects multiple organs and systems and causes significant morbidity and mortality (1). Recently, several criteria have been developed to diagnose SLE or determine SLE activity at an early phase (2). However, owing to its pathogenesis and the exact etiology that has not been completely elucidated, to discover novel biomarkers for early diagnosis of this disease and predicting the therapeutic outcome is very urgent, which enables clinicians to treat SLE patients with the most optimally biologic therapy as early as possible. MicroRNAs (miRNAs) are a class of non-coding small RNAs approximately having 19-25 nucleotides. miRNAs play key roles in regulating post-transcriptional gene expression by complementary pairing to their target messenger RNAs (3,4). Over the last decade, published studies have provided strong evidence for a connection between expression of dysregulated miRNAs and development of several systemic autoimmune diseases, including SLE, which gives us new insights into the pathogenesis of SLE and a new opportunity to find novel diagnostic or therapeutic targets (5)(6)(7)(8)(9)(10)(11)(12)(13). In addition, miRNAs were present in serum or plasma in a remarkably stable form, and prevented degradation from endogenous RNase activity (14)(15)(16)(17). Thus, cell-free circulating miRNAs will be important biomarkers for SLE diagnosis.
It has been demonstrated that uncontrolled over-activated B cells through their maturation into antibody-producing plasma cells are central to the pathogenesis and development of SLE (9,18). B cells also regulate T-cell activity and immune response by acting as antigen-presenting cells or via the production of co-stimulatory molecules and proinflammatory cytokines. Therefore, B cell-related miRNAs may be an attractive target for SLE diagnosis and sequent evaluation of therapeutic outcome.
We have previously screened 72-miRNAs profiling differently expressed in B cells of SLE patients using Affymetrix miRNA 2.0 array (19). In this study, expression of 42 selected miRNAs in a large set of plasma specimens from patients with SLE and rheumatoid arthritis (RA) as well as from healthy controls (HCs) were analyzed using qRT-PCR aiming to identify the miRNAs that could potentially serve as novel serum-based biomarkers for the diagnosis of SLE and further distinguishment between disease activity and lupus nephritis (LN).  (20,21). Disease activity was assessed by the Systematic Lupus Activity Measure and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) at the time of plasma collection. The SLE patients with SLEDAI >15 were polled together and set as highly active SLE group, those SLE patients with SLEDAI <4 were polled and set as stable SLE group. Modification of diet in renal disease formula was used to estimate glomerular filtration rate (eGFR). All the patient's clinical features and treatment drugs were collected: drugs for SLE include steroids, mycophenolate mofetil, cyclophosphamide, azathioprine, MTX, tacrolimus, cyclosporine A, and Leflunomide.

Participants and study Design
The study was designed as a three-phase epidemiological approach to identify the novel circulating miRNAs associated with the disease status of SLE (Figure 1). (1) 10 plasma samples randomly selected from the enrolled SLE patients and 10 plasmas from the age-and sex-matched HCs were used to screen SLE-specific circulating miRNAs from 42 SLE B cell-associated miRNAs using customized miRNAs qPCR array. (2) Different expression of candidate circulating miRNAs was confirmed in the plasma samples comprised of 50 SLE patients, 20 HCs, and

Preparation of Plasma samples and rna isolation
Blood samples were collected into EDTA-2K containing tubes. Blood cells were removed by a two-step centrifugation protocol performed at 4°C (3,000 rpm for 10 min, then 12,000 rpm for 10 min) to obtain platelet-poor, cell-free plasma. All samples were stored at −80°C until analyzed. Plasma RNA was isolated using Trizol ® LS Reagent (Ambion, US) and Direct-zol™ RNA kit (Cat#2060&2062, Zymo Research, US) according to the manufacturer's protocol. To normalize possible sample-to-sample variation caused by RNA isolation, 1 nmol (total volume of 1 µl) of synthetic C. elegans miRNAs cel-miR-39 (MIMAT0000010, Seq: 5′-UCACCGGGUGUAAAUCAGCUUG-3′, GenePharma, China) was added to each denatured sample. The isolated RNAs were digested by Dnase I cocktail to remove the residual DNA, and then were collected in 25 μl DNase/RNase-free water using Direct-zol™ RNA kit. Concentration of RNA was measured by NanoDrop 2000 (Thermo Fisher scientific, US). Isolated RNA was kept at −80°C or immediately used for reverse transcription.
screening of B cell-related mirnas in sle Plasma Using the customized qrT-Pcr mirna array Two independent experiments were performed to detect the different expressions of B cell-related miRNAs in SLE plasma using the customized miRNAs array (CT Bioscience, Jiangsu, China). To eliminate the difference among different samples as well as possible, five plasma samples from SLE patients or HC were mixed as one pool. The panel of the customized miRNA array contained 42 B cell-related miRNAs selected from our previous study (19). First, aliquot 100 ng of plasma RNA was added the poly A tail and then transcribed into cDNA according to the manufacturer's protocol. qRT-PCR detection using the customer PCR chip contained 42 selected miRNA panels was performed on an Applied BioSystems 7500 Real-Time PCR system (Life Technologies). Data were automatically analyzed with SDS Relative Quantification Software version 2.2.2 (Life Technologies). U6, let-7d and let-7g were set as internal controls for plasma miRNA (22), and cel-miR-39 was set as the external control for plasma RNA isolation. The miRNAs that the Ct values were greater than 35 or with unqualified dissolution curve were removed from all datasets. Average Ct values of the duplicate analysis of each miRNA were then subtracted from the average Ct value of the cel-miR-39 or the internal control of U6, let-7d and let-7g for that particular sample, yielding the ΔCt values. The relative expression level of each miRNA was measured through the equation 2 −ΔCt . The experiment was repeated three times.

Detection of mirnas expression
Plasma miRNA expressions of 119 samples from the First Affiliated Hospital of Wenzhou Medical University were detected. First, 100 ng plasma RNA was reversely transcribed using miScript II RT kit (Qiagen, Valencia, German) according to the manufacturer's protocol. Stem-loop Real-time qPCR was performed using the miScript SYBR Green PCR Kit (Cat#218073, Qiagen, German) according to the manufacturer's protocol. The expressions of Based on the results of SLE-specific circulating miRNA, we investigated whether miRNAs could be used as a new diagnostic marker of SLE using the ROC curve. As shown in Figure 3 and    Figure 3 and Table 4, respectively. The diagnostic sensitivity for SLE difference from healthy people was 0.61~0.97 and the specificity was 0.61~1.   Figure 4 and We next investigated the relationship between miRNAs expressions in plasma and established clinical variables of SLE patients ( Table 6). There were only several miRNAs associated with some anti-nuclear antibodies although 14 selected miRNAs were SLE B cell-associated miRNAs. The expression levels of miR-19b, miR-22, miR-23a, and miR-25 in SLE patients with positive anti-β2GP1 were significantly higher than negative anti-β2GP1 patients (P < 0.05). The expression level of miR-15b was significantly higher in SLE patients with positive anti-SSB than those with negative anti-SSB (P = 0.014). There was no correlation between the expression of other miRNA and anti-U1RNP, anti-histone, anti-SSA, anti-Sm, and anti-Ribp in the plasma of SLE. Analysis of the relationship between expression of miRNAs in plasma and disease activity of SLE patients revealed that four miRNAs (miR-15b, miR-93, miR-25, and miR-19b) in the active group were significantly different from the stable group. Expressions of these four miRNAs in active state of SLE were all significantly lower than those in the stable SLE group (Figure 5A). According to the analysis of ROC curve (Figure 5B) (Figure 4; Table 5). The diagnostic sensitivity for SLE distinguish from RA patients was 0.59~0.87 and the specificity was 0.71~0.94.  in SLE patients with low eGFR (eGFR < 60 ml/min/1.73 m 2 ) than those with better renal function (eGFR > 60 ml/ min/1.73 m 2 ) (P = 0.01 and 0.012, respectively). Analysis of ROC curve showed that the AUCs for miR-15b and miR-22 in determining SLE patients with renal damage (eGFR < 60 ml/ min/1.73 m 2 ) were 0.872 (95% CI: 0.714-1.000) and 0.859 (95% CI: 0.677-1.000), with sensitivity of 0.800 or 1.000 and specificity of 0.800 or 0.600, respectively (Figure 6). It did not have any significant association between expressions of plasma miRNAs and other SLE clinical variables ( Table 7), except for miR-23a, miR-25, miR-16, miR-15b, miR-150, and miR-223. miR-23a was associated with gender, miR-25 was associated with CRP, and miR-15b, miR-150, and miR-223 were associated with ALT. Expression of plasma miR-16 was significantly lower in recurrent SLE patients than in SLE patients with first onset (P = 0.045). Analysis of treatment drugs showed that all SLE patients used steroids and 28 patients used immunosuppressive drugs among 50 SLE patients.

Double Validation of mir-15b as a Biomarker for sle Patients With Disease activity and ln
Based on the above analysis, miR-15b could act as a potential disease biomarker for SLE patients with disease activity and LN with low eGFR. Thus, to validate the results, we collected 69 SLE samples to further determine the expression of miR-15b. Results showed that miR-15b in the expression of SLE activity group was significant different from the stable group (P = 0.039, Figure 7A). According to the analysis of ROC curve (Figure 7B), miR-15b that separate active and stable state of the SLE patients with an AUC of the ROC curve of 0.665 (95% CI: 0.519-0.797), which determined the sensitivity (0.810) and specificity (0.604). Comparison of plasma miRNA expression level in patients with renal function revealed that the miR-15b in patients with low eGFR (eGFR < 60 ml/min/1.73 m 2 ) was significantly decreased (P = 0.033); the analysis of ROC curve (Figure 7C) showed miR-15b to determine the AUC was 0.820 (95% CI: 0.662-0.978), which determined the sensitivity (0.609) and specificity (1.000).
In addition, results obtained from another 80 SLE samples also showed that miR-15b in the expression of SLE activity group was significantly different from it in stable group (P = 0.01, Figure 7D). ROC curve analysis showed that AUC of miR-15b in predicting activity of the SLE patients was 0.696 (95% CI: 0.560-0.832), which determined the sensitivity at 0.767 and specificity at 0.586 ( Figure 7E). Comparison plasma miRNA expression in patients with different eGFR level, miR-15b in patients with low eGFR (eGFR < 60 ml/min/1.73 m 2 ) was significantly decreased (P = 0.028); based on the analysis of ROC curve, AUC of miR-15b in predicting LN with low GFR was 0.675 (95% CI: 0.508-0.843), which determined the sensitivity at 0.878 and specificity at 0.438 ( Figure 7F). The results of treatment drugs showed that in the cohort of 69/80 SLE patients from Wenzhou/Shanghai, 67/77 patients used steroids and 46/61 patients used immunosuppressive drugs.

DiscUssiOn
MicroRNA is very stable in blood circulation, and can be preserved for a long time without degradation in vitro, which makes miRNA very suitable for using as a new serum marker for a variety of diseases (11). The stability can be partially explained by the discovery of lipoprotein complexes, including small membrane vesicles of endocytic origin (called exosomes or microvesicles), high-density lipoprotein, and circulating Argonaute 2 complexes (23)(24)(25). Here, we used an ROC curve to evaluate the results and found that 14 miRNAs with clinical diagnostic value in the diagnosis of SLE are different from the HCs, six miRNAs with clinical diagnostic value are different between SLE and RA patients. The differential expression of miRNAs in plasma of SLE patients might be used as new biomarkers. Moreover, we analyzed the correlation between SLE-specific circulating miRNA and clinical features of patients with SLE, including autoantibodies and other related indicators. Results indicated that expressions of miR-19b, miR-25, miR-93, and miR-15b in SLE activity group were significantly lower than those in the SLE stable group. In addition, further analysis indicated that expressions of miR-15b and miR-22 correlated significantly with renal damage. Further diagnostic potential of miR-15b for SLE disease activity and LN was selected and validated on an independent validation set with 69 SLE patients and a cross-validation set with 80 SLE patients. Circulating miRNA has been reported to be differentially expressed in SLE patients, but data are inconsistent in different studies, we reviewed them in PubMed and collected together  in Table 8. Carlsen et al. screened 45 plasma miRNAs in two independent cohorts using quantitative RT-PCR assays (26). They found that seven miRNAs were statistically significant in plasma from SLE patients. Expression of miR-142-3p and miR-181a was increased while expression of miR-106a, miR-17, miR-20a, miR-203, and miR-92a was decreased. A 4-miRNA signature was diagnostic of SLE, and a specific subset of miRNA profiles was associated with nephritis. Wang et al. screened the plasma miRNA profiles of SLE patients by miRNA microarrays and identified eight miRNAs by RT-qPCR (27). They found that circulating levels of miR-16, miR-223, miR-23a, miR-15b, miR-150, and miR-25 were upregulated in patients with SLE in contrast to that expressions of circulating miR-155 and miR-92a were downregulated. Surprisingly, SLE-associated miR-155 was not significantly dysregulated in the plasma of both SLE and RA patients, compared with HCs. Using miRNA PCR array, Kim et al. found that nine miRNAs were differentially expressed between the SLE and HC (28). Three miRNAs (hsa-miR-30e-5p, hsa-miR-92a-3p, and hsa-miR-223-3p) were significantly upregulated in plasma of SLE patients (P = 0.048, P = 0.039, and P = 0.046, respectively). In our study, we found the plasma levels of 14 miRNAs were downregulated. The change of miR-92a in patients with SLE was consistent with previous studies (26,27 Recently, several published studies have shown that miR-15b is involved in the pathologies of autoimmune disease. Liu et al. identified miR-15b as an important factor in Th17-associated effects and determined that expression of miR-15b was significantly downregulated in multiple sclerosis patients and in mice with experimental autoimmune encephalomyelitis (33). They also found that O-linked N-acetylglucosamine transferase is a potential target of miR-15b, enabling it to affect the transcriptional regulation of retinoic acid-related orphan receptor γT through O-linked N-acetylglucosamine glycosylation of NF-κB. Singh et al. reported that miR-15b/16 enhances the induction of regulatory T cells by regulating the expression of Rictor and mammalian target of rapamycin (34). Ren et al. used a B-cell profiling chip analysis and found that CyclinD3 was related to SLE and significantly elevated in SLE B cells (35). Furthermore, they demonstrated that activation of TLR7 dramatically increased CyclinD3 expression but significantly decreased miR-15b in B cells in vitro. They identified further that CyclinD3 is a direct target of miR-15b. Our results also showed that plasma miR-15b was decreased in patients with SLE, and was positively correlated with disease activity and LN with low eGFR. Thus, a larger sample size including different pathological types of LN, in a well-designed study will be promising for understanding of the functional role of miR-15b in future.
In conclusion, we have demonstrated here that differential expression of circulating miRNAs is a typical character between SLE patients and HCs. The plasma miR-15b correlated with SLE activity and LN may serve as a biomarker for SLE.

DaTa aVailaBiliTY sTaTeMenTs
All relevant data are contained within the manuscript, all datasets for this study are included in the manuscript and the  aUThOr cOnTriBUTiOns HZ, XH, and LY: performed the experiments, analyzed and interpreted the data, and drafted the manuscript. GG: performed the experiments and statistical analysis. XL, CC, LS, and BL: acquired the data and material support. XX and NC: made contribution to the conception and design, analyzed and interpreted the data, supervised the study, provided the project funding, revised the manuscript, and finally approved the version of the manuscript for publication.
supplementary files. All the raw data of this manuscript are available by the authors, without undue reservation, to any qualified researcher.

acKnOWleDgMenTs
The authors thank all the patients and their families, physicians, and technicians who were involved in the study.

FUnDing
This work is supported by grants from the Zhejiang Provincial Natural Science Foundation of China (LY12H05003 and LY14H050006), the Zhejiang Science and Technology Bureau (2012C33126,2016C33214), and the Wenzhou Science and Technology Bureau (Y20170744). These sponsors provide the funding for the experiments and the collection of specimens.