Methylation And Expression Of GLIPR1 Gene In Acute Myelogenous Leukemia

Background and Objective: Leukemia is the common hematopoietic malignancy that results from an imbalance between cell proliferation and cell maturation,and the development and progression of leukemia is a multi-factor and multi-step process that involved in genetic and epigenetic changes. In recent years, the study found DNA methylation is high frequent events in the human leukemia, which is closely related to its pathogenesis. The methylation of promoter region CpG islands is one of main ways for inactivating tumor suppressor gene, DNA repair gene and pro-apoptotic gene. The methylation-silenced genes not only play an important role in the pathogenesis of leukemia, but also provide a specific target for the treatment of leukemia. GLIPR1 gene is a new gene cloned from the human glioblastoma cell line, and functions as a tumor suppressor gene in the human prostate cancer. Gingras etc. found that the expression of GLIPR1 increase during the differentiation and maturation of the human monocytes in 2000 year, and can serve as a marker for myeloid monocyte differentiation, indicating that GLIPR1 may be down-regulated in acute myeloid leukemia (AML) and be involved in its pathogenesis. But till now GLIPR1's expression and regulatory mechanism in AML is unclear. Thus, in this study the levels of GLIPR1 mRNA and protein expression level and methylation status in the AML were detected to explore if GLIPR1 is methylation-silenced gene in AML.Methods: 5 leukemia cell lines, 70 cases of bone marrow from the newly diagnosed AML patients, 57cases of bone marrow from the acute lymphoblastic leukemia (ALL) patients, 40 cases of bone marrow from the chronic myeloid leukemia (CML) patients, 125 cases of control bone marrow, and 24 cases of bone marrow from the complete remission AML patients were enrolled for the current study. RT-PCR and Western blotting were used to detect the mRNA expression and protein levels of GLIPR1, methylation-PCR (MS-PCR) was performed to detect the promoter methylation status of GLIPR1, and the relationship between the mRNA and protein expression levels and methylation status in GLIPR1 gene was analyzed.Results: (1) The mRNA expression level of GLIPR1 in the AML cell lines was lower than that in the CML and ALL cell lines, whereas the methylation level of GLIPR1 in the former was higher than that in the latter. The mRNA expression level of GLIPR1 in the AML cell lines was significantly increased, but had not obvious changes in the CML and ALL cell lines after 5-aza-2dC treatment. The methylation level of GLIPR1 in the AML cell lines was obviously lower than that in the ALL and CML cell lines after 5-aza-2dC treatment; (2) The average mRNA expression level of GLIPR1 in the AML bone marrows was obviously lower than that in the ALL(0.38±0.20 VS 0.76±0.18), CML(0.38±0.20 VS 0.80±0.14) and control bone (0.38±0.20 VS 0.85±0.12)marrows, and in the bone marrows with complete remission AML was obvious higher than that in the AML bone marrows before treatment(0.78±0.13 VS 0.36±0.20), but there was not a significant difference between the ALL and CML bone marrows and control bone marrows(0.76±0.18VS 0.85±0.12, 0.80±0.14VS 0.85±0.12). (3) The average protein expression level of GLIPR1 in the AML bone marrows was obviously lower than that in the ALL(0.14±0.05VS 0.32±0.12), CML(0.14±0.05 VS 0.36±0.16) and control bone (0.14±0.05 VS 0.40±0.08) marrows, and in the bone marrows with complete remission AML was obvious higher than that in the AML bone marrows before treatment (0.38±0.16 VS 0.16±0.07), but t there was not a significant difference between the ALL and CML bone marrows and control bone marrows (0.32±0.12 VS 0.40±0.08, 0.36±0.16 VS 0.40±0.08). (4) The positive rate of GLIPR1 gene methylation in the AML bone marrows was obviously higher than that in the ALL(82.8% VS 38.6%), CML(82.8% VS 27.5%) and control bone (82.8% VS 16.8%) marrows, and in the bone marrows with complete remission AML was obviously lower than that in the bone marrows before treatment (12.5% VS 75%), but there was not a significant difference between the ALL and CML bone marrows and control bone marrows (38.6% VS 16.8%, 27.5% VS 16.8%). (5) There was a negative relationship between the expression levels of mRNA and protein and methylation status of GLIPR1 in the AML bone marrows.Conclusion: (1) There is high frequent methylation of GLIPR1 gene with expression down-regulation in AML, and the methylation of GLIPR1 gene promoter may be a reason for GLIPR1 gene silence, which indicate that GLIPR1 gene is a methylation-silenced gene in the AML. (2) The methylation level of GLIPR1 gene may have utility for evaluating the AML patients' curative effect.