MicroRNA-3148 modulates allelic expression of toll-like receptor 7 variant associated with systemic lupus erythematosus

Abstract

We previously reported that the G allele of rs3853839 at 3'untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10(-10), odds ratio (OR) (95%CI) = 1.27 (1.17-1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10(-11), OR = 1.24 [1.18-1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3'UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R(2) = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3'UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10(-19), OR = 1.25 [1.20-1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.

Figure 1. Allelic associations of SNPs in the TLR7-TLR8 region with SLE.

(A) The genomic structure of the TLR7-TLR8 region and the location of all studied SNPs are indicated. (B) Association signals (−log₁₀ P) are plotted against the position of each SNP (based on GRch37/hg19) in European Americans (EA), African Americans (AA), and Hispanics (HS). Genotyped and imputed SNPs are depicted with circles and triangles, respectively. The diamond identifies the TLR7 3′UTR SNP rs3853839. SNPs are highlighted using different colors according to their LD strength (r²) with rs3853839. (C) A trans-ancestral meta-analysis is conducted on 40 genotyped SNPs (circles) and 14 imputed SNPs (triangles) that are shared by the three ancestries (SNPs listed in Table S1) using fixed and random model, respectively. The dashed line represents the significance level of 5×10⁻⁸.

Figure 2. The SLE-risk G allele of rs3853839 confers elevated TLR7 expression through slower mRNA degradation.

(A) Association of rs3853839 genotypes with TLR7/8 transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter in vitro. TLR7 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in TLR7 transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in TLR7 transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. ^* P<0.05. (E) Summary of the G/C allele ratio in TLR7 transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; P = 0.04; paired t test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.

Figure 3. The SLE-risk G allele of rs3853839 displays reduced transcript modulation by miR-3148.

(A) TargetScan's predicted miR-3148-binding site in TLR7 3′UTR. The C allele, rather than G allele of rs3853839 corresponds to the second base of this seed region. (B) Inverse correlation of miR-3148 and TLR7 transcript levels in PBMCs from 16 patients with SLE (solid circles) and 21 controls (open diamonds). (C) HEK-293 cells were cotransfected with empty reporter vector (EV), luciferase constructs driven by TLR7 3′UTR segment containing either C or G allele of rs3853839 and increasing concentrations (1, 6, 12, and 48 nM) of miR-3148 or nontarget control (NC) mimics. Luciferase activity was determined 24 hours after transfection. Normalized luciferase activity is the Renilla/Firefly ratio of miR-3148-treated reporter vector compared with the same NC-treated reporter vector. Data show the mean ± SEM and are representative of cumulative data from three independent experiments. P = 0.0003 over all miR-3148-treated C-allele vector groups, and not significant over all miR-3148-treated G-allele or empty vector groups (ANOVA test). P* = 0.02, P**<0.0001 (Student's t test) for the comparison of indicated groups.