The Effect Of DNA Methylation On The Expression Of Human TIG2 Gene In Leukemia Cells

Objective: More and more researches show that aberrant DNA methylation status, especially the missing expression of hypermethylation of tumor suppressor gene, may play an important role in leukemogenesis, diagnosis and prognosis of leukemia, detection of minimal residual disease (MRD), as well as guidance for lekemia therapy.Tazarotene is a analogues of retinoic acid and has been used for the treatmnet of skin diseases. TIG1(tazarotene-induced gene-1), TIG2 and TIG3 genes expressed high level in endothelial cells after these cells had been treated with Tazarotene. TIG2 gene is consistently expressed in human normal skin cells,but silenced or low expressed in psoriatic patients. Several studies indicated that TIG2 is a natural ligand of orphan G protein-coupled receptor(GPCR) ChemR23 and is able to recruit immature dendritic cells (DCs) and macrophages (Mφ) in vitro. TIG2 could be activated via the cleavage of C terminal of precursor TIG2 by proteolytic proteasea from serum and inflammation site. TIG2 gene is ubiquitously expressed in human normal tissues,and several studies showed TIG2 gene was expressed in normal lung cells but silenced in lung cancer cells,which indicated that TIG2 gene may play an role in lung tumorigenesis.However, there was no report on the molecular mechanism of silented expression of TIG2 and the expression level of TIG2 gene in hematopoietic tumors. Therefor, the present study was designed to detect TIG2 gene expression in leukemia cells and normal hematopoietic cells, to observe the effect of demethylating agent on TIG2 expression in leukemia cells, to analyze Cp G islands distribution within TIG2 promoter and first extron region in lekemia cell lines and fresh leukemia cells by MethPrime software.Methods:1 Cell cultureTo thaw leukemia cell lines (K562, U937, KG1), resuspend the cells in RPMI 1640 and culture the cells in a 5.0% CO2 containing incubator, at 37℃.2 To collect and preserve bone marrow mononuclear cells (BMMNCs)Bone marrow was collected from leukemia patients and health volunteers. Mononuclear cells (MNCs) was extracted from bone marrow for the next step.3 Total RNA was extracted from BMMNCs of leukemia patients or health volunteers. The expression level of TIG2 mRNA was detected by Real-Time Quantitative PCR.4 To forecast the Cp G island within promoter region of TIG2 geneTo identify methylation sites,we designed the methylated primer(M) and unmethylated primer(U) for MSP and analyzed Cp G islands distribution within TIG2 promoter and first extron region by MethPrimer software.5 The expression level of TIG2 mRNA was detected by Real-Time Quantitative PCR in leukemia cell lines after these cells treated with demethylation agent 5-Aza-CdR.6 To detect the methylation status of the Cp G island of TIG2 geneGenomic DNA, extracted from leukemia cell lines and MNC of lekemia patients, was transformed with sulfite and purificated, and then the incidence of methylation within Cp G islands of TIG2 gene was examed by MSP.Results:1 The expression level of TIG2 gene in leukemia cells and normal hematopoietic cellsWe found that TIG2 gene expressed at high level in normal BMMNCs,but silenced or at low level in leukemia cells. (β-actin was used as an inner reference).2 The expression level of TIG2 gene in leukemia cells after treated with demethylation agent.Leukemia cell lines K562, U937, KG1 were treated with demethylating agent(5-Aza-CdR) at different concentrations. After the treatment, TIG2 gene expression were restored, and effect of 5-Aza-CdR was in a dose-dependent manner.3 The Cp G island within promoter region of TIG2 geneWe found there were typical Cp G island within the promoter and the first exon region of TIG2 gene.4 Detection of methylation status of the Cp G island in TIG2 geneThe promoter methylation status of TIG2 gene was examined and found that TIG2 gene was unmethylated in normal control samples,but was frequently methylated in leukemia cells and leukemia cell lines.Conclusion:1 TIG2 gene expressed at high level in normal hematopoietic cells, but silenced or expressed at low level in leukemia cell;2 After leukemia cells treated with demethylation agent 5-Aza-CdR, TIG2 gene expression in leukemia cells were restored, and the effect of 5-Aza-CdR was in a dose-dependent manner.3 Aberrant methylation of promoter of TIG2 gene was frequently present in leukemia cell lines and leukemia samples and methylation may be responsablefor the decreased transcription of TIG2 gene.