| Objective:The research of promoter methylation before was mainly focused on mono-promoter gene. But the gene regulation of multiple promoters is more complex than that of mono-promoter. Research of methylation of estrogen receptor (ER) with four promoters was mainly focused on steroid-related cancers. This experiment was designed to disclose the methylation status of ER promoters and clarify the significance of ER promoters methylation in silencing ER expression in leukemia. Analysis the possiblility of ER Promotor serve as epigenetic biomarkers in the diagnosis, prognosis and treatment prediction of leukemias. The correlations of ER methylation , ER expression and ER protein expression were further studied which provided theory basis for the relation between the epigentic and the pathogenesis of various cancers and new direction for the therapy of cancers.Methods:1. The leukemia cell line culture and treatment with 5-Aza CdR , including Jurkat, HL-60, K562, U937, THP-1 and Molt-4.2. Using methylation specific PCR (MSP) and direct DNA sequencing to detect the CpG island methylation changes of ERa-A, -B,–C and ERβpromoters in the leukemia cell lines before and after 5-Aza CdR treatments3. Using RT-PCR to detect the expressions of ERa-A, -B,–C and ERβbefore and after 5-Aza CdR treatment.4. Using Western-Blot to detect the protein expressions of ERa and ERβbefore and after 5-Aza CdR treatment.5. The cell apoptosis, cycle, proliferation and viability with and without 5-Aza CdR were evaluated with 3H-thymidine (3H-TdR) incorporation, Propidium Iodide (Pi) simple staining and typan blue staining. 6. the methylation and expression of ERs was further analyzed in acute leukemia patients and health control by MSP and RT-PCR.7. We tracked 40 cases of leukaekimas with ERα-A methylation (95%; 38/40) to observe the prognosis with one year's chemotherapy.Results:1. MSP analysis showed CpG islands of ERa-A, -B,–C and ERβpromoters were methylated and ERa-A mRNA expressions were inactivated in the six leukemia lines,including HL-60,K562,Jurkat,Molt-4,U937.2. With 5-Aza CdR treatment , CpG islands of ERa-A, -B,–C and ERβpromoters were demethylated and ERa-A mRNA were re-expressed.3. With 5-Aza CdR treatment , the protein expression of ERa was enhanced significantly, compared with protein expression of ERa without 5-Aza CdR treatment (p<0.01)4. With 5-Aza CdR treatment , the cycle, proliferation and viability of leukemia cell lines were all inhibited significantly, compared with that without 5-Aza CdR treatment (p<0.01)5. With 5-Aza CdR treatment , the apoptosis of leukemia cell lines was increased significantly, compared with that without 5-Aza CdR treatment (p<0.01).6. To further evaluate the methylation status and expression activity of ER promoter in leukemia, RT-PCR and MSP was performed to analyze the promoter expression and CpG methylation of ERa isoforms (i.e., ERa-A, -B, -C) and ERβin 40 cases of leukemia patients which consisted of 12 cases of ALL, 21 cases of AML, and 7 cases of CML. Compared with full-unmethylation in all healthy controlss (100%; 10/10), ERa-A promoter was semi-methylated and its expression was inactivated in almost all of leukemia patients (95%; 38/40), including 92% (11/12) in ALL, 95% (20/21) in AML, and 100% (7/7) in an CML. Compared with full-methylation of ERa-A promoter, the other two isoforms of ERa, i.e., ERa-B and -C, showed as full- and semi-methylation in healthy controls, respectively. And there were no apparent methylation patterns of both ERa-B and -C in leukemia patients except for both of them were methylated. The ERβshowed as fully methylated in all patients (100%, 40/40) and healthy controls.7. The patients with ERa-A methylation have no obvious symptomatic relief, however, two patients without ERa-A methylation have obtained effective reliefConclusions:1. Two isoforms of progesterone receptor, the CpG islands of ERa-A, -B, -C and ERβpromoters were hypermethylated in leukemia cell lines. However, only mRNA expression of ERa-A was inactived.2. With 5-Aza CdR treatment, the CpG islands of ERa-A, -B, -C and ERβpromoters were demethylated. Furthermore, ERa-A mRNA was restored in all cell lines, and there were no changes in the expression of ERa- B, -C, and ERβ. suggesting that inactivation of ERa-A was caused by methylation in leukemia.3. Western-blot was performed to analyze the expression of ERa and ERβon the leukemia cell lines with or without 5-aza-dC demethylation treatment. Without treatment of 5-aza-dC, the protein expression of ERa were deficient in all leukaemia cell lines including Jurkat, HL-60, K562, U937, THP-1, and Molt-4. However, with the DNA demethylation treatment of 5-aza-dC, only the expression of ERa was significantly enhanced in all cell lines, and there were no obvious changes in the expression of ERβ.4. 3H-TdR incorporation, Propidium Iodide (Pi) simple staining and typan blue staining were performed to analyze the cell apoptosis, cycle, proliferation and viability on the leukaemia cell lines with or without 5-aza-dC demethylation treatment. With the DNA demethylation treatment of 5-aza-dC, the cell apoptosis, cycle, proliferation and viability have significant changes, compared with leukaemia cell lines without 5-aza-dC demethylation treatment(p<0.01).5. The CpG islands of ERa-A promoter were unmethylated in healthy samples, but ERa-A hypermethylation in leukemia samples (95%). This fact indicates that ERa-A promoter methylation may be a potential marker for leukemia diagnosis.6. the methylation status of ERa-A CpG island could provid theory for the selection of Endocrine therapy and prognosis judgment...
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