Functional Assessment of HLA-E Promoter and 3' UTR Polymorphisms

Advances in sequencing technology have enabled extensive identification of polymorphisms in the regulatory regions of the HLA-E gene. However, the regulatory mechanisms of gene expression for most polymorphic sites remain unclear, limiting research on the associations between HLA-E genetic variation and disease. In this study, we investigated the regulatory potential of two polymorphic sites in HLA-E: NT-26 G/T in the promoter (rs76971248) and NT + 3345 T/C in the 3' UTR (rs376446052) (nucleotide numbering according to the IPD-IMGT/HLA Database v3.55 released January 2024). Bioinformatics tools were used to predict transcription factors and miRNAs that may bind to these polymorphic regions. Dual-luciferase reporter assays were performed to validate the interactions between the HLA-E promoter and transcription factor HOXA5, as well as between the 3' UTR and miR-619-5p. Additionally, TaqMan probe assays were conducted to analyse the frequency of NT-26 polymorphism in healthy individuals and solid tumour patients. Bioinformatics analysis predicted a stronger binding affinity of HOXA5 to the mutant HLA-E promoter (NT-26 T). Dual-luciferase assays confirmed that the NT-26 T variant significantly reduced basal promoter activity compared to the wild-type (NT-26 G) (p < 0.0001). Furthermore, HOXA5 binding induced a more pronounced suppression of transcriptional activity in the mutant promoter (p < 0.01). For the 3' UTR, miR-619-5p significantly inhibited the transcriptional activity of both the wild-type and mutant (NT + 3345 C) constructs (p < 0.05), with no differential effect observed between the two variants (p > 0.05). Genotyping analysis revealed that the frequency of the NT-26 T allele did not differ significantly between solid tumour patients and healthy controls (p > 0.05), contrasting with its previously reported lower frequency in haematological malignancies. In conclusion, the HLA-E regulatory polymorphism NT-26 functionally modulates gene expression by altering HOXA5-mediated transcriptional repression, contributing to a disease-specific association pattern, particularly in haematological malignancies. In contrast, the NT + 3345 polymorphism does not influence miR-619-5p-dependent regulation. Suggesting that this particular 3' UTR variant may lack a direct functional role in miRNA-mediated control of HLA-E expression. These findings elucidate the mechanistic roles of HLA-E regulatory variants and provide a foundation for understanding their impact on disease susceptibility.