| Objective To measure methylation level of SFRP gene in the cell lines of nine malignant hematonosis and primary or relapsed acute leukemia (AL) patients by DNA sequencing and Methylation Specific PCR (MSP), and then the screened hypermethylated cell line was used to study the mechanisms that how 5-aza-2'-deoxycytidine (5-Aza-CdR) would reverse hypermethylation of SFRP and inhibition effect on proliferation in the acute leukemia cell line-Jurkat.Methods DNA sequencing and MSP were used to screen the methylation level of cell lines and AL patients. After the target cell line was identificated, CCK-8 methods were used to evaluate the effect that 5-aza-2'-deoxycytidine had on the ability of cell reduplication and cell vitality; Both methylation levels of Jurkat before and after 5-aza-2'-deoxycytidine disposal were analyzed by MSP; The mRNA expression level of DNA methyltransferase 1 (DNMT1), DNMT3A, DNMT3B andβ-catenin, survivin, c-myc, cyclin-D1 were analyzed by semi-quantiative reverse transcription-polymerase chain reaction (RT-PCR); The expression of SFRP detected by Real time fluorescence quantiative RT-PCR, Hoechst-PI and Annexin V FITC/PI were used to estimate the apoptosis in each group, Western-blotting assay was used to estimate the protein level ofβ-catenin.Results (1) Hypermethylation of SFRP1,2 genes was present in nine malignant hematopoiesis cell lines, however, methylation and unmethylation of SFRP4 were both detected in CA46, HL60 and U937 cell lines, and SFRP5 in U266 as well; None of the normal mononuclear cells showed methylation of SFRP genes; The frequencies of aberrant methylation among the patient samples were 33.9%(20/59) for SFRP1, 23.7%(14/59) for SFRP2,6.8%(4/59) for SFRP4 and 10.2%(6/59) for SFRP5 in AML, and 39.3%(11/28) for SFRP1,28.6%(8/28) for SFRP2,25.0%(7/28) for SFRP4 and 32.1%(9/28) for SFRP5 in ALL. (2) SFRP gene in Jurkat was found hypermethylated and is a choice option for pharmaceutical study on demethylation; The methylation level was apparently attenuated after 72h disposal of 5-aza-2'-deoxycytidine, and hypermethylation of SFRP had been successfully reversed. (3) Compared with the untreated group, after 72h disposal of 5-aza-2'-deoxycytidine, the expression of DNMT1, DNMT3A and DNMT3B was obviously down-regulated in a concentration-dependent manner. (4) 5-aza-2'-deoxycytidine significantly inhibited the proliferation of Jurkat and induced cell apoptosis; compared with the untreated group, after the treatment of 5-aza-2'-deoxycytidine, the protein level ofβ-catenin was reduced; mRNAs of the downstream target genes survivin, c-myc and cyclin-D1 were decreased.Conclusion (1) Hypermethylation of SFRP genes is a common early event in the evolution of AL, Methylation of SFRP genes might serve as potential independent biomarkers for early detection of AL. (2) 5-aza-2'-deoxycytidine could activate the SFRP gene by demethylation. (3) The mechanisms that 5-aza-2'-deoxycytidine reversed hypermethylation of SFRP or/and by inhibting DNMT1, DNMT3A and DNMT3B genes. (4) 5-aza-2'-deoxycytidine could significantly depress the proliferation rate of Juekat, which may be through down-regulation the Wnt/β-catenin sigaling pathyway.
|
PDF Full Text Request