P061 : PROMOTER METHYLATION CAN AFFECT THE ANTIGEN AND mRNA EXPRESSION OF KIR3DL1

Abstract

To determine the effects of promoter methylation on the expression of KIR3DL1, we assessed promoter methylation patterns of NK cell line (NK-92), normal NK cells and myelogenous leukemia cell line (K562). The KIR3DL1 genotypes were analyzed by PCR-SSP and PCR-SBT. The promoter methylation patterns were detected by bisulfite sequencing technique. The expressions of KIR3DL1 were determined by flow cytometry and real-time quantitative PCR (RQ-PCR). The genotype was 3DL1 ∗ 001/3DL1 ∗ 002 for NK-92 and 3DL1 ∗ 00501 homozygote for K562. Among five chosen blood donors, three were 3DL1 ∗ 01502 homozygote and two were 3DL1 ∗ 00501 homozygote. The expression of KIR3DL1 on NK-92, K562 and 3DL1 ∗ 00501 samples were extremely low, but were high on 3DL1 ∗ 01502 samples. Treatment of NK-92 with 5-Aza significantly increased the expression of KIR3DL1 on cell surface ( Fig. 1 ), but the K562 did not increase evidently. The core promoter region of NK-92 and three 3DL1 ∗ 01502 samples exhibited 19 CpG islands, but NK-92 exhibited a higher methylation at +1 CpG position compared with 3DL1 ∗ 01502 samples. The NK-92 and three 3DL1 ∗ 01502 samples all composed of high expression alleles ( ∗ 001, ∗ 002 and ∗ 01502), which implied that +1 CpG position may be crucial in 3DL1 expression. K562 and two 3DL1 ∗ 00501 samples had 17 CpG islands, while these three samples did not express 3DL1. RQ-PCR showed that 5-Aza treatment greatly increased the transcription of NK-92, but did not affect the K562. The KIR3DL1 antigen and RNA expression in NK-92 cells is greatly affected by methylation, and the +1 CpG position may be important for expression. We conclude that KIR expression are mainly epigenetically determined and maintained through DNA methylation.