Zhongguo shi yan xue ye xue za zhi · 2017
[Effect of AML1-ETO Fusion Protein on Transcriptional Regulation of p14ARF].
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Zhuang Wen-Yue, Li Zheng-Yi, Chen Yi-Hua, Liu Yan, Chen Zi-Xing
Аннотация
OBJECTIVE: To investigate the effect of transcriptional regulation of aberrant transcription factor AML1-ETO on p14ARF.
METHODS: P14ARF expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. Methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of p14ARF promoter. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and p14ARF promoter in AML1-ETO positive leukemia cell line. And the p14ARF mRNA expression level was detected by qRT-PCR after treatment with 5-Aza.
RESULTS: AML1-ETO-expressing cell subclone displayed low level of p14ARF mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, level of p14ARF mRNA was markedly lower when compared with other acute myeloid leukemias lacking this translocation. P14ARF gene promoter was non-methylated in control group and primary leukemia cells of AML patients without t(8;21) and was hyper-methylated in U937-A/E1-4 and primary leukemia cells of AML patients with t(8;21). The enriched regions in transfected cells were located within p14ARF promoter. 5-Aza could increase the expression of p14ARF.
CONCLUSION: P14ARF is a possible target gene of AML1-ETO. The p14ARF silencing induced by hyper-methlylation may be an important factor for occurrence and development of the M2b subtype of acute myeloid leukemia.
METHODS: P14ARF expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. Methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of p14ARF promoter. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and p14ARF promoter in AML1-ETO positive leukemia cell line. And the p14ARF mRNA expression level was detected by qRT-PCR after treatment with 5-Aza.
RESULTS: AML1-ETO-expressing cell subclone displayed low level of p14ARF mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, level of p14ARF mRNA was markedly lower when compared with other acute myeloid leukemias lacking this translocation. P14ARF gene promoter was non-methylated in control group and primary leukemia cells of AML patients without t(8;21) and was hyper-methylated in U937-A/E1-4 and primary leukemia cells of AML patients with t(8;21). The enriched regions in transfected cells were located within p14ARF promoter. 5-Aza could increase the expression of p14ARF.
CONCLUSION: P14ARF is a possible target gene of AML1-ETO. The p14ARF silencing induced by hyper-methlylation may be an important factor for occurrence and development of the M2b subtype of acute myeloid leukemia.