| Background and aim:DNA Methylation occurs frequently in early cancer. Hypermethylation leads to gene silencing, which is involved in tumor genesis and development. Non-Hodgkin's lymphoma is a hematological malignancy, whose clinical treatment has step into the era of molecular targeted therapy. However, its stage evaluation and prognostic monitor are still in the stage of imageology and cell morphology, which is dropping behind of therapy. Professor Li Yu et al. found that Zonula occludens-1(ZO-1) gene may be associated with hematologic malignancies using the screening method of Restriction Landmark Genomic Scanning (RLGS), and the abnormal methylation in its promoter region plays an important role in these diseases. ZO-1 is a tight junction protein, involved in maintaining the permeability of barrier next to the normal cell, cell signal transduction, gene transcription and regulation of cell proliferation and differentiation, which suggested that it is closely related to the differentiation and invasion of cancer. Preliminary studies have confirmed that the ZO-1 gene was epigenetically silenced by DNA methylation in human leukemia cell line HL60 and natural killer cell line NK92-MI, suggested that it may be a tumor suppressor gene in hematological malignancies. This study aims to ivestigate the methylation status of ZO-1 gene in non-Hodgkin's lymphoma and its relationship with stage, type, remission, and explore its significance as a genetic marker for clinical diagnosis.This study was divided into two parts.Part I:Methods:Using methylation specific-PCR (MS-PCR) to detect the methylation status of the ZO-1 gene promoter region in 29 cases of paraffin embedded tissues of non-Hodgkin's lymphoma (NHL). Results:(1) Specimens were detected in 29 cases,17 cases had methylation bands,2 cases had completely non-methylated bands, and 10 cases did not appear amplification. The methylation rate was 89.4% (17/19) in NHL, which was significantly higher than that in reactive hyperplasia lymph node.(p< 0.05). (2) 4 cases of T-cell NHL, the methylation rate was 100% (4/4),15 cases of B-cell NHL, the methylation rate was 86.6%(13/15), (P> 0.05). (3) Specimens were detected in 39 patients,26 patients got the band, the detection rate was 66.7%, of which there are 16 cases more than 2 years specimens, access to test results in 7 cases, the methylation rate was 43.7%; 1-2 years specimens in 7 cases, test results obtained in 3 cases, the methylation rate was 42.9%; 1 year 16 cases of specimens, all obtained results, more than 2 years, threre was no significant difference between 1-2 samples (P> 0.05), and there was significant difference in the methylation rate with 1 year samples(P<0.05). Conclusion:1, The methylation rate of ZO-1 gene promoter region in NHL tissues were significantly higher than that in reactive hyperplasia lymph node, there was little difference between the various subtypes. The methylation of ZO-1 promotor region can serve as a general gene marker for secondary diagnosis of NHL; 2, Because the methylation rate of specimens declined with the preparation time, using fresh tissue may increase the detection sensitivity and lower the false negative rate.Part IIMethods:Detection of the methylation status of ZO-1 gene promoter in of the bone marrow sample from 45 patients with sub-NHL by MS-PCR.These samples included 5 cases of idiopathic thrombocytopenic purpura (ITP) and 5 cases of normal bone marrow samples. Results:(1) The methylation status of ZO-1 gene promoter region in the bone marrow:the methylation rate of ZO-1 gene was 53.85%(21/39) in patients with newly diagnosed or relapsed or non-complete remission of NHL patients.There was significant difference between NHL patients, ITP patients and healthy donors.(P<0.05); (2) The complete remmision(CR) group were statistically different with initial treatment, recurrence group and the non-CR group, while there was no significant difference between non-CR, initial treatment, and recurrence group. (3) There was no significant difference in the methylation status of ZO-1 gene promoter region between various types (P> 0.05).The follicular cells of B cells various subtypes had highest methylation rate, followed by diffuse large B cell lymphoma, marginal zone lymphoma, mantle cell lymphoma, and finally, Burkitt lymphoma,. (4) In 39 NHL patients who were newly diagnosed, relapsed or complete remission, there was 18 cases with ZO-1 gene methylation, and the positive rate was 64.29%(18/28). In 11 NHL patients at stage I, II, there was 3 cases with ZO-1 promotor gene methylation, and the positive rate was 27.27%, p<(0.05). (5) Case analysis showed that 2 cases of newly diagnosed patients at stage IV with ZO-1 promotor methylation became methylation-negative after CR. Conclusion:The hypermethylation status of ZO-1 gene promoter region was associated with the disease stage, remissionl, which is a prospective adjunctive marker to evaluate the progress and prognosis and to guide the clinical treatment of NHL.In summary, the methylation status of ZO-1 gene promoter was different at different stage of the disease.There was no difference among different pathological types., It is a prospective universal marker to aid the molecular diagnosis, evaluate the stage and guide the therapy, which was expected to be a new helpful basis in genetic markers, staging, guiding therapy.
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