Functional Assessment of HLA ‐E Promoter and 3′ UTR Polymorphisms

ABSTRACT

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.