Single-nucleotide polymorphisms in 3'-untranslated region inducible costimulator gene and the important roles of miRNA in alopecia areata

Abstract

Background: Alopecia areata (AA) spares the stem cell compartment and attacks only the base of the hair follicle, which is surrounded by infiltrating lymphocytes. AA is associated with polymorphisms in immune-related genes and with decreased function of CD4+CD25+ T regulatory (Treg) cells. Treg function is modulated by the costimulatory molecules, like inducible costimulator (ICOS) that are crucial in orienting T cell differentiation and function so that they strongly impact on the immunologic decision between tolerance or autoimmunity development.

Objective: The aim of our study was to investigate the possible association of AA with single-nucleotide polymorphisms (SNP) present in the ICOS 3'-untranslated region (3'UTR) region and to elucidate how SNPs modulate ICOS gene expression by affecting miRNA binding sites.

Methods: This is a case-control study performed in 184 patients with AA and 200 controls. ICOS gene and miRNA expression were analyzed by real-time polymerase chain reaction.

Results: The genotype carrying the rs4404254(C) [p = 0.012, OR (95% CI): 0.5 (0.3-0.8)] and rs4675379(C) [p = 0.015, OR (95% CI): 0.3 (0.1-0.8)] 3' UTR alleles was more frequently observed in AA patients than in controls and correlated with a reduced ICOS expression. miR-1276 significantly suppressed ICOS expression by binding to the 3'UTR of ICOS mRNA. Also, we observed that, miR-101 and miR-27b are upregulated, while miR-103 and miR-2355-3p are downregulated in peripheral blood mononuclear cells of AA patients compared to controls.

Conclusion: Our data show that rs4404254 and rs4675379 SNPs of ICOS gene are associated with AA and also reveal that the presence of rs4404254 polymorphism correlates with ICOS post-transcriptional repression by microRNA binding.

FIGURE 1

The risk allele of rs4404254 is associated with lower inducible costimulator (ICOS) mRNA expression. (a) Relative expression measurements of ICOS expression were calculated by real‐time PCR. Fold change (y‐axis) represents the relative expression of ICOS in 30 AA patients (AA pts) in comparison to 30 healthy controls (ctrl) (p = 0.0004). (b) Correlation analysis between the SNP genotypes and ICOS expression in 12 AA patients carrying the rs4404254 risk allele, and 18 patients carrying the rs4404254 non‐risk allele. For the rs4404254 risk allele we considered the C/C and C/T genotypes, and for the non‐risk allele the T/T genotype (p = 0.01). Error bars in (a) and (b) represent mean and SD of three independent experiments. Normalization of the expression data for the ICOS gene was realized with GAPDH using the 2^{‐∆∆ Ct} method formula

FIGURE 2

Relative expression of miRNA in PBMCs from healthy subjects and AA patients. Relative expression measurements of miRNA were calculated by real‐time PCR, using the 2^{‐∆∆ Ct} method formula. Fold change (y‐axis) represents the relative expression of the miR‐103, miR‐27b, miR‐369, miR‐101, miR‐1276 and miR‐2355‐3p genes in AA patients (circular plots) in comparison to healthy controls (rectangular plots). U6 small nuclear RNA was used as a control to determine relative miRNA expression. Error bars represent mean and SD of three independent experiments

FIGURE 3

Schematic representation of the construct used in the luciferase assays. A fragment of 760 bp of the ICOS 3′UTR, encompassing rs4404254 wild‐type allele (a) or rs4404254 allelic variant (c) the putative responsive elements for miR‐1276 or encompassing rs4675379 wild type (b) or rs4675379 allelic variant (d), the putative responsive elements for miR‐2355‐3p, was cloned in pGL3 vector downstream to the Firefly luciferase coding sequence. Nucleotides in miRNA sequence paired with ICOS 3′UTR are shown in capital letters and miRNA nucleotide paired with the corresponding allelic variant of ICOS 3′UTR SNPs is underlined in bold

FIGURE 4

miR‐1276 and miR‐2355‐3p target the ICOS 3′UTR. 293T cells were transfected independently with pGL3‐Report luciferase plasmid or pICOS‐3′UTR vector together with either specific microRNA miR‐1276 or miR‐2355‐3p. Forty‐eight hours after transfection, the percentage of cells expressing the protein luciferase was measured by flow cytometry and normalized to the Firefly control. The results represent the mean and standard error (SE) of at least three independent experiments, each carried out in triplicate. The risk allele for rs4404254 SNP was (C) allelic variant (SNP1 mut) and the non‐risk allele was (T) allelic variant (SNP1 wt), while for rs4675379 SNP the risk allele for was (C) allelic variant (SNP2 mut), and the non‐risk allele was (G) allelic variant (SNP2 wt)