Interaction of miR-181b and IFNA1 Polymorphisms on the Risk of Systemic Lupus Erythematosus

Introduction A previous work has discovered that chromosome 1q32 locus linked to the risk of systemic lupus erythematosus (SLE) and miR-181b located on the susceptibility site with downregulation inversely correlating to its target molecular interferon alpha 1 (IFNA1). The purpose of this study was to investigate the association of miR-181b and IFNA1 polymorphisms with IS risk. Methods The miR-181b rs322931, IFNA1 rs1332190, and rs10811543 were genotyped using a Multiplex SNaPshot assay. miR-181b expression levels in plasma of SLE patients and controls were analyzed using quantitative PCR. Results The rs322931 CT, CT/TT, and T allele exerted an increased trend of SLE risk (CT vs. CC: adjusted OR = 1.71, 95% CI 1.16-2.50, P = 0.01; CT/TT vs. CC: adjusted OR = 1.45, 95% CI 1.08-1.95, P = 0.01; T vs. C: adjusted OR = 1.38, 95% CI 1.07-1.79, P = 0.01). Combined genotypes of the rs322931 CT/TT+rs1332190 TT and the rs322931 CC+rs10811543 AG/AA also revealed an increased risk of SLE. Gene-gene interaction analysis showed that a three-locus model consisting of rs322931, rs1332190, and rs10811543 attributed an increased risk of SLE. Further genotype-phenotype analysis revealed that rs322931 CT/TT carriers displayed lower levels of miR-181b. Conclusions These findings indicate that the miR-181b rs322931 may be singly and jointly responsible for the etiology of SLE by altering miR-181b expression.

In addition to coding genes, noncoding transcripts, such as microRNAs (miRNAs) also play a critical role in modulating immune response of SLE [13][14][15][16]. Until today, more than 1500 miRNAs have been sequenced in human [17], with an estimation of regulating over one-third of genome expression [18]. Among them, the miR-181 family was found to be downregulated in patients with SLE, being an important modulator of B and T cell differentiation and inflammatory reaction, which were key events in the initiation and development of SLE [13,[19][20][21][22].
Recently, miRNA-related genetic variant has been reported to be a risk factor for a series of human diseases, including SLE [23][24][25][26][27][28][29][30]. For example, an SNP rs4937333 T allele was reported to be associated with a significantly increased risk of SLE by enhancing the binding of miR-5003 to transcriptional factor ETS1 and decreasing ETS1 expression [26]. A genetic variant of rs322931 was reported to alter both transcriptional activity and the expression of miR-181b [29,30]. To date, no study reported the association of miR-181b rs322931 with the risk of SLE. In this hospitalbased case-control study, we investigated whether the miR-181b rs322931 was related to the occurrence of SLE in a Chinese Han population. As miR-181b may contribute to the development and progression of SLE through directly targeting molecular interferon alpha 1 (IFNA1) [13], two SNPs (i.e., rs1332190 and rs10811543) in the promoter region of IFNA1 were also examined for gene-gene interaction analysis. We found that the miR-181b rs322931 and IFNA1 rs1332190 and rs10811543 may play an interactive role in the etiology of SLE.  [31]. The controls were also registered from the same hospital at the same period. We excluded those controls if they had autoimmune diseases, such as malar butterfly rash, photosensitivity, fever, erythra, and edema. All the subjects without blood transfusion were genetically unrelated ethnic Han Chinese living in Guangxi province. After written informed consent was signed, each participant donated 3-5 mL of eathylene diamine tetraacetic acid-anticoagulated peripheral venous blood. Plasma was separated by centrifuging for 10 min at 1000 rpm and stored in -80°C until analysis. The study protocol was approved by the Institutional Review Board of the Affiliated Hospital of Guilin Medical University.

SNP Selection.
We selected miR-181b rs322931 locating on the chromosome 1q32 that has been identified to be a risk locus of SLE [32]. Additionally, we selected SNPs within the IFNA1 that is a target gene of miR-181b [32], with the inclusion criteria of minor allele frequency > 10% in Chinese Han population. Two SNPs (i.e., rs1332190 and rs10811543) in the promoter of IFNA1 were selected.

Genotyping.
Genomic DNA was extracted from leukocytes of peripheral blood using the commercially available kit (Tiangen, Beijing, China). The miR-181b rs322931 and IFNA1 rs1332190 and rs10811543 were genotyped using a Multiplex SNaPshot assay. For quality control, negative control replacing template DNA with distilled water was used in each run and about 5% of all samples were randomly selected for validation using DNA sequencing. The results between the two genotyping methods were concordant.
2.4. Quantitative PCR (qPCR) of miR-181b. Total RNA was isolated from plasma using a commercial kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. cDNA was generated using the Mir-X miRNA First-Strand Synthesis Kit (Takara Bio USA, Mountain View, CA, USA) according to the manufacturer's manual. miR-181b levels in cases and controls were quantified using the Mir-X miRNA qRT-PCR TB Green Kit (Takara Bio USA). qPCR was run on an ABI 7900HT real-time PCR machine (Applied Biosystems, CA, USA). U6 was used as an internal control, and relative expression of miR-181b was determined using the 2 -ΔΔCt method [33].
2.5. Statistical Analysis. Statistical analysis was done using SPSS software version 13.0 (SPSS, Chicago, IL, USA). The χ 2 test was used to evaluate Hardy-Weinberg equilibrium (HWE) and the differences of the rs322931, rs1332190, and rs10811543 between cases and controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the association between each SNP and SLE risk using a logistic regression model after adjustment for age and gender. Linkage disequilibrium (LD) and haplotype analysis were performed using the SHEsis software [34]. The statistical significant value of SNPs was set as 5 × 10 −8 that was used in genome-wide association study. Gene-gene interaction analysis was performed by using multifactor dimensionality reduction (MDR) platform [35]. miR-181b expression levels were compared using the Mann-Whitney U test, and a value of P < 0:05 was considered statistically significant.

Characteristics of Study Population.
The characteristics of the study population are summarized in Table 1. There were no significant differences in the distribution of age and gender (P = 0:27 and 0.10, respectively). Approximately half of the SLE patients had photosensitivity, leucopenia, anaemia, complement depression, renal disorder, and arthritis, while only about 30% patients had malar rash and negative AdsA. Most of the patients (87.3%) had positive ANA.

Main Effects of SNPs in miR-181b and IFNA1 on SLE
Risk. The genotype and allelic frequencies of the selected SNPs among cases and controls are presented in Table 2. The genotype distributions of the miR-181b rs322931 and IFNA1 rs1332190 and rs10811543 met the HWE requirements in both cases and controls (P > 0:05). Compared to the miR-181b rs322931 CC genotype, individuals with the CT variant genotype exerted an increased trend of SLE risk (adjusted OR = 1:71, 95% CI 1.16-2.50, P = 0:01). Under the dominant genetic of inheritance, carriers with the CT/TT genotypes had a 1.45-fold increased risk of SLE (adjusted OR = 1:45, 95% CI 1.08-1.95, P = 0:01). Moreover, carriers with the T allele had a 1.38-fold increased risk of SLE (adjusted OR = 1:38, 95% CI 1.07-1.79, P = 0:01). For the rs1332190 and rs10811543, no significant difference between SLE patients and controls was found (P > 0:05). Stratification analysis also showed null association between the three SNPs and clinical features of SLE (Table 3).

Haplotype and Combined
Analysis. LD results revealed that the rs1332190 and rs10811543 were in strong LD (D ′ = 0:97, r 2 = 0:67). The frequencies of 4 possible haplotypes are shown in Table 4. There was no significant difference of the haplotype between cases and controls.

Interaction Analysis.
3.5. The rs322931 CT/TT Exhibited Lower Levels of miR-181b. The expression levels of miR-181b were analyzed using qPCR in controls (n = 44) and SLE patients (n = 42). As shown in Figure 1(a), the miR-181b expression was lower in SLE patients compared to controls (P < 0:05). When comparing the relationship of the rs322931 to miR-181b expression, we found that the rs322931 CT/TT carriers had lower levels of miR-181b than rs322931 CC carriers (P < 0:05) (Figures 1(b) and 1(c)).

Discussion
In this hospital-based case-control study, we found that the miR-181b rs322931 C but not IFNA1 rs1332190 and rs10811543 was associated with an increased trend of SLE risk, whereas combined analysis showed a significant association of rs322931-rs1332190 and rs322931-rs10811543 with SLE risk. Gene-gene interaction analysis also showed that     BioMed Research International the three-locus model consisting of rs322931, rs1332190, and rs10811543 attributed an increased risk of SLE. In addition, genotype-phenotype analysis revealed that the rs322931 CT/TT carriers displayed lower levels of miR-181b. Our data shed light on the importance of miR-181b rs322931 in the setting of SLE. The miR-181 family including miR-181a, miR-181b, miR-181c, and miR-181d is a critical regulator in the pathogenesis of SLE [13-16, 19, 36, 37]. Altered expression of miR-181a was not only observed in SLE and lupus nephritis patients but also correlated to clinical features, such as the erythrocyte sedimentation rate, C reactive protein, anti-dsDNA antibody, complements, and score of SLE disease activity index [14-16, 36, 37]. miR-181b was also reported to be differentially expressed in SLE patients [13]. These findings indicate that miR-181 may be a biomarker for the diagnosis of SLE and monitor of SLE activity.
Previously, an SNP rs322931 was found to be a risk factor for positive emotion, reward processing, and ischaemic stroke through influencing transcriptional activity and the expression levels of miR-181b [29,30,38]. The current work has discovered that chromosome 1q32 locus linked to the risk of SLE and miR-181b located on the susceptibility site with the downregulation inversely correlating to its target molecular IFNA1 [32]. Based on this background, we hypothesized that the rs322931 may affect individual's susceptibility to SLE. Our findings in this association study confirmed this hypothesis. We found that the rs322931 CT/TT genotypes and T allele carriers had a 1.45-fold and 1.38-fold increased risk of SLE, respectively. qPCR was then performed to detect the expression of miR-181b, and we found that the miR-181b levels were significantly lower in SLE patients than controls, confirming previous report [32]. Of note, after comparing the correlation of the rs322931 to miR-181b expression, we found that carriers with the rs322931 CT/TT genotypes exhibited lower levels of miR-181b in both SLE patients and controls. Taken together, we may conclude that the rs322931 CT/TT genotypes and T allele contributed to the risk of SLE by decreasing the levels of miR-181b.
In addition to the miR-181b rs322931, we examined much more SNPs in this study because SLE is a complex disease and involved in more than one gene. Genome-wide association study has provided evidence of susceptibility loci of SLE in the interferon (IFN) signaling pathway [39]. IFN-α is highly expressed and has emerged as a key pathogenic cytokine in SLE [40,41]. For example, IFN-α in sera from active SLE patients can induce differentiation of dendritic cells that capture and present antigens to CD4+ T cells [42]. IFN-α also exerts stimulatory effects on the adaptive immune system by enhancing B cell differentiation and survival of autoimmune B cells [43,44]. Moreover, IFN-α impairs autophagic degradation of mtDNA and vasculogenesis in SLE, serving as a drug target [45,46]. IFN-α is encoded by IFNA1 that is a target gene of miR-181b [13]. Genetic polymorphisms in IFNA1, therefore, were analyzed in this study. Although no significant association of the rs1332190 and rs10811543 in the promoter of IFNA1 with SLE risk was observed in single site analysis, combined analysis revealed an association of the 2 SNPs with SLE occurrence. Both the rs322931 CT/TT+rs1332190 CT/CC and the rs322931 CC+rs10811543 AG/AA were associated with a reduced risk of SLE. Finally, we performed miR-181b/IFNA1 interaction analysis, and we found that the miR-181b rs322931/rs1332190/rs10811543 was the best candidate model with the accuracy of 0.67. Our findings indicate that the miR-181b rs322931 may be singly and jointly responsible for the etiology of SLE.
Some limitations should be discussed in the interpretation of our data. It is evident that nongenetic factors such as cigarette smoking, oxidative stress, ultraviolet light, infection, and hormonal action play important roles in the development of SLE [3][4][5][6]. In this study, we did not collect these factors and thus, the association of miR-181b and IFNA1 polymorphisms with environment factors cannot be analyzed. Additionally, the samples in this study were not large enough and all of them were enrolled from Han Chinese. Further studies with larger sample sizes are required to assess whether our positive findings can be confirmed in other ethnicities.

BioMed Research International
In summary, we found for the first time that the rs322931 C risk allele was related to the development of SLE and the rs322931 CT/TT genotypes altered miR-181b expression levels in SLE patients. Of note, miR-181b-IFNA1 interaction conferred the risk of SLE. Once confirmed in other ethnicities with larger sample sizes, it is likely to be important for future personalized treatment of SLE by genotyping the miR-181b rs322931 and IFNA1 rs1332190 and rs10811543.

Data Availability
The digital data used to support the findings of this study are available from the corresponding author upon request.

Additional Points
Key-points. 1. Individuals with the miR-181b rs322931 CT, CT/TT, and T allele had an increased trend of SLE risk. 2. Combined analysis showed a significant association of rs322931-rs1332190 and rs322931-rs10811543 with SLE risk. 3. Gene-gene interaction analysis showed that the three-locus model consisting of rs322931, rs1332190, and rs10811543 attributed an increased risk of SLE. 4. The rs322931 CT/TT carriers displayed lower levels of miR-181b.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Review Board of the Affiliated Hospital of Guilin Medical University (2012022) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent
Informed consent was obtained from all individual participants included in the study.