Regulation of KIR3DL3 Expression via Mirna.

Rungtiwa Nutalai,Silvana Gaudieri,Amonrat Jumnainsong,Chanvit Leelayuwat

doi:10.3390/genes10080603

Abstract

Killer-cell immunoglobulin-like receptor (KIR) 3DL3 is a framework gene present in all human KIR haplotypes. Although the structure of KIR3DL3 is suggestive of an inhibitory receptor, the function of KIR3DL3 has not been demonstrated and cognate ligands have not been identified. KIR3DL3 has been shown to be constitutively expressed at a low RNA level in peripheral blood mononuclear cell (PBMC) and decidual natural kill (NK) cells, but cell surface expression of KIR3DL3 cannot be detected. Accordingly, post-transcriptional regulation of KIR3DL3 should exist. Using bioinformatics analysis, we identified three candidate micro ribonucleic acids (miRNAs; miR-26a-5p, -26b-5p and -185-5p) that potentially regulate KIR3DL3 expression. Luciferase reporter assays utilizing constructs with mutated miRNA-binding sites of miR-26a-5p, -26b-5p and -185-5p in the 3’-untranslated region (3’ UTR) of KIR3DL3 resulted in up-regulation of luciferase activity demonstrating a potential mechanism of gene regulation. Furthermore, knockdown of the same endogenous miRNAs using silencing ribonucleic acid (siRNA) led to induced surface expression of KIR3DL3. In conclusion, we provide a novel mechanism of functional regulation of KIR3DL3 via miRNAs. These findings are relevant in understanding the generation of KIR repertoire and NK cell clonality.

Highlights

Killer-cell immunoglobulin-like receptors (KIRs) are transmembrane glycoproteins consisting of both inhibitory and activating receptors on natural kill (NK) cells and on a subset of T cells [1,2].NK cells exhibit at least one inhibitory receptor to discriminate cells with aberrant human leucocyte antigen (HLA) class I surface expression often observed during infection and malignant transformation from normal cells [3]
In agreement with previous reports [31], six micro ribonucleic acids (miRNAs) were matched with the list of miRNAs expressed in human (CD56+ CD3- ) NK cells, namely miR-26a-5p, -26b-5p, -185-5p, -203a-3p, -372 and miR-373
To determine whether these miRNAs could contribute to the regulation of KIR3DL3 at the post-transcriptional step, NK92 and YT cells were analyzed for the expression of the six candidate miRNAs by Stem-loop Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

Summary

Introduction

NK cells exhibit at least one inhibitory receptor to discriminate cells with aberrant human leucocyte antigen (HLA) class I surface expression often observed during infection and malignant transformation from normal cells [3]. This receptor-ligand interaction is important in establishing the NK cell activation threshold and is influenced by the diversity in both sets of molecules. Four conserved KIR genes are present in virtually all individuals, known as the framework genes KIR3DL3, KIR3DP1, KIR2DL4, and KIR3DL2 [6,7], but only KIR2DL4 is expressed at both the RNA and protein

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