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To test the hypothesis that methylation of a CpG island is associated with regulation of microRNA expression, we investigated CpG islands in the upstream sequences of microRNA precursors (pre-miRNAs) through bioinformatic analysis and determined whether the CpG islands were methylated by methylation-specific PCR in the k-562 cell line. We used 5-azacytidine for DNA demethylation, and changes in microRNA expression were detected by microarray assay, RT-PCR, and real-time PCR after 5-azacytidine induction. We showed that the CpG islands in the upstream regions of 18 pre-miRNAs were methylated, including miR-663, miR-369, miR-615, and miR-410, and promoter activity was detected in the upstream region of pre-miR-663. We found that a decrease in methylation of a CpG island could up-regulate the expression of miR-663, suggesting that miR-663 could be regulated by DNA methylation. Expression levels of miR-369, miR-615, and miR-410 were not regulated by DNA methylation in this cell line.

This study was purposed to investigate the difference of zo-1 gene promoter methylation between healthy individuals and acute leukemia patients. BS-PCR method was used to detect the status of zo-1 gene methylation in healthy individuals, acute leukemia patients and leukemic cell line NB4 cells. The results showed that zo-1 gene was hypomethylated in bone marrow samples from healthy individuals (1.9%). In newly diagnosed AL and relapsed patients, the rate of zo-1 gene methylation was 93.2% and 66.9% respectively, while it was 16.4% in AL patients in complete remission, which was much higher than that in healthy individuals. There was significant difference between them. It is concluded that as compared with healthy individuals, zo-1 gene in acute leukemia patients is hypermethylated and with different degrees in various phases of leukemia. Analysis of zo-1 gene methylation status may be useful to monitor the development of acute leukemia.

In order to investigate the effect of demethylating treatment on the expression of inhibitory KIR and the cytolytic activity of NK-92MI cells, and to study the possible relationship between the demethylation of inhibitory kir gene and the function of NK cells. NK-92MI cells were treated with 5-azacytidine to induce DNA demethylation. The expression of KIR3DL1, KIR2DL2/KIR2DL3, KIR2DL1 and the viability of NK-92MI cells were detected by flow cytometry. The KIR3DL1 positive and the KIR3DL1 negative NK-92MI cells were also sorted by flow cytometry. The cytotoxicity of NK-92MI against K562 cells was detected by the LDH release assay. The results demonstrated that the expressions of KIR3DL1, KIR2DL2/KIR2DL3 and KIR2DL1 in NK-92MI cells all increased after treating with 1.0, 2.5 and 5 micromol/L of 5-azacytidine for 72 hours. And the cytotoxicity of NK-92MI against K562 cells decreased. In these dose range, 5-azacytidine did not influence the viability of NK-92MI cells. Additionally, the cytotoxicity of KIR3DL1 positive NK-92MI cells was lower than that of the KIR3DL1 negative cells. It is concluded that the demethylating treatment suppresses the cytotoxicity of NK-92MI cells through increasing the expression of inhibitory KIR.

The aim of this study was to analyze the promoter methylation patterns of inhibitory killer cell immunoglobulin-like receptor (KIR) which gene expression and the effect of demethylation treatment were studied, and to explore the possible regulation mechanism of inhibitory kir gene expression. The promoter methylation levels of kir2DL1 and kir2DL2/kir2DL3 in NK-92MI cell line were detected by bisulfite sequencing technique. Then NK-92MI cells were treated with 5-azacytidine to induce the demethylation of CpG islands. The levels of gene expression of kir were determined by RT-PCR. The results demonstrated that the methylation frequencies of CpG dinucleotides surrounding the promoter regions of kir2DL1 and kir2DL2/kir2DL3 genes were 25% to 88% and 5% to 80% respectively. DNA-demethylating treatment with 5-azacytidine resulted in re-expression of kir2DL1 gene and increased expressions of kir2DL1, kir2DL2 and kir2DL3 genes in NK-92MI cells. In conclusion, the promoter DNA methylation participates in the regulation of kir gene expression in NK-92MI cells.

Aim: To investigate the methylation pattern of KIR3DL1 promoter region in NK cell line NK-92MI and the effect of demethylation and histone acetylation on gene expression, and to study the possible regulation mechanism of KIR3DL1 expression., Methods: The methylation status of KIR3DL1 promoter in NK-92MI cells was detected by bisulfite sequencing technique. Then NK-92MI cells were treated with the DNA-demethylating compound 5-azacytidine and (or) the inhibitor of histone deacetylase trichostatin A, and the expression of KIR3DL1 gene was observed., Results: The CpG dinucleotides surrounding promoter region of KIR3DL1 gene in NK-92MI cells were consistently methylated with a frequency of 70%-100%. After 72 h treatment with 2.5 micromol/L and 5 micromol/L of 5-azacytidine, the mRNA expression of KIR3DL1 gene in NK-92MI cells increased 66.6% and 114.6%, respectively. However, after 72 h treatment with 50 nmol/L of trichostatin A, the mRNA expression of KIR3DL1 gene in NK-92MI cells did not increase. Additionally, combined treatment with 5-azacytidine and trichostatin A did not lead to a synergistic effect compared with 5-azacytidine alone., Conclusion: KIR3DL1 expression in NK-92MI cells is regulated by promoter methylation.

To analyze the methylation pattern of kir3dl1 promoter and its relationship with gene expression, and to study the possible regulation mechanism of kir3dl1 gene expression, the methylation status of kir3dl1 promoter in K562 cell line was detected by bisulfite sequencing technique. Then K562 cells were treated with 5-azacytidine so as to induce de-methylation of CpG island, and the relationship of CpG island methylation with kir3dl1 gene expression was investigated. The results demonstrated that CpG dinucleotides surrounding core promoter region of kir3dl1 gene was methylated at a frequency of 20% to 100% in K562 cell line. Comparison of promoter sequence with GenBank database revealed a base substitution within a putative binding site for the transcription factor E2F in K562 cell line. This mutation forms a new methylation site in kir3dl1 promoter. DNA-demethylating treatment resulted in de novo expression of kir3dl1 gene in formerly non-expressed K562 cell line. It is concluded that kir3dl1 expression in K562 cells is regulated by DNA methylation. Deeply to study E2F function contributes to profound understanding of its role in kir3dl1 gene expression regulation.

Abstract: NK cells as a component of the innate immune system provide a first line of defense against viral infections and malignancies. Killer immunoglobulin-like receptors (KIRs) are a polymorphic gene family expressed on NK cells. The crucial role of DNA methylation in determining the variegated expression pattern of KIRs has recently been reported. In this study the direct effect of DNA demethylating treatment on human NK cell cytotoxicity was analyzed. In a human NK cell line NK-92MI, inhibitory KIR genes exhibited characteristic epigenetic repression, whose promoter regions are densely methylated. Treatment of NK-92MI cells with methyltransferase inhibitor 5-azacytidine significantly increased the expression levels of inhibitory KIRs and strongly suppressed their cytolytic activity against human K562 leukemic cells. Cytolytic activity of human polyclonal NK cells was also suppressed upon 5-azacytidine-treatment. The suppression of NK cell cytotoxicity was associated with 5-azacytidine-induced overexpression of inhibitory KIRs and impaired granzyme B and perforin release by these cells. The results demonstrate for the first time that demethylating treatment can suppress NK cell cytolytic activity. The aberrant methylation patterns of KIR genes during NK cell differentiation and maturation may have importance for its abnormal function. [Copyright &y& Elsevier]

Abstract: The KIR3DL1 gene is a member of killer immunoglobulin-like receptors family, which exhibits a variegated expression pattern in NK cells and subsets of CD4+ and CD8+ T cells. The E2F family of transcription factors plays a crucial role in the regulation of gene expression. The present study reports a naturally occurring point mutation (TTTGGCGC→TTCGGCGC) within a putative E2F binding site in the KIR3DL1 promoter in K562 cells. Interestingly, this mutation introduces a new methylation site. This study shows for the first time that E2F1 binds to the KIR3DL1 promoter in vivo. This point mutation and concomitantly altered methylation pattern within the E2F1 binding site abolishes their binding and reduces the promoter activity, while elevated expression of E2F1 correlates with increased promoter activity. Therefore, E2F1 contributes to the transcriptional activation of the KIR3DL1 gene. [Copyright &y& Elsevier]