Background/Purpose:

We identified a genome-wide significant association of the TLR7 variant (rs3853839) with SLE susceptibility in multiple ancestries (P_meta =2.0×10^-19, OR =1.25), and the risk allele carriers exhibited elevated levels of TLR7 mRNA and protein and more pronounced type I interferon (IFN) signature in SLE PBMCs. At or near the location of this variant are targeted sequences for predicted multiple microRNA (miRNAs) binding sites that may affect transcript degradation, leading us to explore whether differential miRNA binding could explain allelic risk of TLR7 for SLE.

Methods:

Each of the six bioinformatically predicted miRNAs was co-transfected with the TLR7 3’UTR reporter construct carrying either the risk or non-risk allele of rs3853839 into HEK 293 cells to assess which miRNA inhibits luciferase activity. To test effects of miRNA(s) on regulation of endogenous TLR7 expression and downstream IFN-α production, PBMCs from healthy donors were transfected with miRNAs or non-target control miRNAs for 24 hours, followed by stimulation with TLR7 agonist (CL097) for 24 hours to induce IFN-α production and measured in culture supernatants by ELISA. Cells were lysed for RNA extraction and mRNA levels of TLR7 and IFN scores (MX1, Ly6E, IFIT1 and IFIT3) were quantified by RT-PCR.

Results:

Transfection of each of six miRNAs into HEK-293 cells showed that (1) only miR-3148 and miR-2278 could reduce luciferase activity driven by the TLR7 3’UTR construct containing either risk or non-risk allele of rs3853839; (2) allelic differences in reduction of luciferase activity were observed by overexpression of miR-3148 at increasing concentrations [reduction in non-risk allele vs. risk-allele construct: 13.2% vs. 4.8%, P =0.023 (6nM); 22.5% vs. 9.9%, P =0.0012 (12nM); 21.4% vs. 8.5%, P =0.0031 (48nM)], but only by overexpression of miR-2278 at the highest concentration [31.3% vs. 15.6%, P =0.039 (48nM)]. Expression levels of miR-3148, but not miR-2278, were inversely correlated with TLR7 mRNA levels in PBMCs (R² = 0.255, P =0.001, and R² =0.086, P =0.08, respectively). Compared to the non-target control miRNA, preliminary results of overexpression of miR-3148 in PBMCs (n=15) led to 15% reduction in TLR7 mRNA expression (P =0.003), resulting in a trend of decreased downstream production of IFN-α (P =0.07); whereas inhibition of miR-3148 led to 24% increase in TLR7 mRNA expression (P =0.006) and a trend of increased production of IFN-α (P =0.09). Overexpression or inhibition of miR-3148 in PBMCs showed no significant difference in IFN scores.

Conclusion:

Among the 6 tested miRNAs, miR-3148 and miR-2278 showed allelic difference in degradation of mRNA containing rs3853839, which results in increased expression of TLR7 mRNA in risk-allele carriers. The inverse correlation between miR-3148 and TLR7 mRNA levels and a trend of downregulation of IFN-α production in PBMCs ex vivo support miR-3148 regulates TLR7 expression and affects downstream IFN pathway. Our data suggests interactions between the genetic risk factor (rs3853839) and the epigenetic factor (miRNAs), implicating possibility of miRNA-based therapies to ameliorate SLE where excessive TLR7 activation exists.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microrna-mediated-regulation-explains-allelic-risk-of-tlr7-variant-predisposing-to-systemic-lupus-erythematosis/