Association between polymorphisms in the promoter region of miR‐17‐92 cluster and systemic lupus erythematosus in a Chinese population

Abstract The aim of this study was to investigate the association of genetic polymorphisms in the promoter region of miR‐17‐92 with systemic lupus erythematosus (SLE). The gene polymorphism was analysed using SNaPshot in 312 SLE patients and 396 controls. Relative expression of miR‐17‐92 was measured by quantitative real‐time PCR. Association was found between rs9515692 and a decreased risk of SLE (CT vs CC: OR = 0.65, 95%CI, 0.46‐0.92, P = .014; CT+TT vs CC: OR = 0.64, 95%CI, 0.46‐0.90, P = .009; T vs C: OR = 0.69, 95%CI, 0.52‐0.92, P = .010, respectively). Haplotype analysis showed that C‐G‐G, C‐A‐A haplotypes were associated with an increased SLE risk (OR=4.46, 95%CI, 2.17‐9.17, P < 0.001; OR=2.33, 95%CI, 1.44‐3.76, P < 0.001, respectively). T allele and CT+TT genotypes in rs9515692 were associated with decreased risk of anti‐dsDNA in SLE (CT+TT vs CC: OR = 0.42, 95%CI = 0.24‐0.72, P = .002; T vs A: OR = 0.49, 95%CI = 0.31‐0.79, P = .003). Moreover, rs9515692 CT+TT genotypes had a higher level of miR‐17 as compared to CC genotype (P = .017). These findings suggest that the rs9515692 CT+TT genotypes were a protective factor for the susceptibility of SLE, probably by increasing the expression of miR‐17.

individual susceptibility to SLE. Previous studies have been established that genes have important effects on the development of SLE such as LEP and LEPR gene, FccR gene, miRNA-146a. [3][4][5] Successes from these studies generally explain only part of disease heritability in SLE. Hence, the molecular genetic basis of this disease remained deficiently understood.
MiR-17-92 displays different expression level during B-cell development: they are enhanced level in progenitor cells, and their expression decreases highly when pre-B becomes immature B cells. 6 MiR-17-92 expression is up-regulated in CD4 + T cells from lupus patients and multiple sclerosis. 7,8 Decreased expression of miR-17-92 has been found during differentiation towards CD8 + T cells. 9 It is generally recognized that these immune-related cells were related to the development of SLE.
To date, no report was carried out to investigate the association of SNPs in miR-17-92 and SLE risk, and the relationship between miR-17-92 gene SNPs and the expression of plasma miR-17-92 family members in SLE patients. We use EPD (http://www.epd.isb-sib. ch/) and miRbase (http://www.mirbase.org/) to predictive promoter.
Then, the SNPs of Minor allele frequency (MAF) greater than 10% were selected by UCSC (http://genome.ucsc.edu) in the Chinese population. Therefore, we evaluate the association of the three SNPs (rs9515692 and rs1352743) in the promoter region of miR-17-92 with susceptibility to SLE and further investigate the influence of miR-17-92 polymorphisms on critical plasma levels and various disease clinical features.   Table S2.

| Quantitative PCR of miR-17-92
Total RNA was isolated from 100 lL plasma performed with a commercial kit (Takara, Dalian, China) following the manufacturer's protocol. Five microgram of total RNA was transcribed into cDNA

| Statistical analysis
If the data were normally distributed variables, the Student's t-test was used; otherwise, Mann-Whitney U test was used. Hardy-Weinberg equilibrium (HWE) was tested using chi-squared test. Haplotype analysis was performed using SHEsis software (http://analysis.bio-x. cn/myAnalysis.php) . OR and 95% CI were adjusted based on age and gender using logistic regression. P < .05 was considered statistically significant.

| RESULTS
There was no significant difference between cases and controls in age (P = .087) and gender (P = .124). The distributions of the four SNPs polymorphisms in SLE and controls are shown in Table 1.
All genotype distributions were in agreement with the Hardy-   in SLE patients was compared with healthy controls. This indicated that miR-17 expression in plasma may suppress SLE development.
One of the most characteristic antibodies of SLE is anti-doublestranded DNA (Anti-dsDNA), which as a sensitive symbol in the disease. Overexpression of miRNA17-92 cluster was associated high titres of anti-DNA antibodies. 8 We identified that rs9515692 was related to decreased risk of anti-dsDNA. The finding further provides evidence that rs9515692 may be a protective factor for SLE. The molecular mechanism of how the rs9515692 leads to the up-regulation of miR-17 requires a more detailed analysis.
MiR Decreased expression of pro-apoptotic molecule and phosphatase and tensin homologue on chromosome 10 in mice transgenic for the miR-17-92 cluster, lead to lymphoproliferation and other lupus manifestations. 16 It is generally recognized that these cells play primary mediators in SLE. Given the key roles of miR-17-92 in SLE development, the positive results in our present study were biologically reasonable.
In conclusion, this is the first time reported miR-17-92 gene polymorphisms associated with SLE susceptibility in an independent Chinese cohort. Analyses suggested that the rs9515692 decreased the risk of SLE in the Chinese population. Therefore, our findings may provide new insights into the development of SLE and create an opportunity to approach the diagnosis and treatment. In the future, further functional studies of rs9515692 will help us to define the potential biological mechanism of SLE.