Study On P73 Gene Methylation And Demethylation In Non-Hodkin's Lymphomas

Objectives: Non-Hodkin's Lymphomas(NHL) is a kind of malignant hematologic tumors that threaten people's health and life severely. At present, the pathogenesis of NHL is not well understood, and its main therapy is combined chemo-therapy. Chemo-therapy has severe ill effects on bone marrow and other organs that affect the life quality of patients and limit the clinic therapeutic efficacy. Our experiment is objected to explore the pathogenesis of NHL, especially the role of p73 gene CpG island methylation on occurrence and development of NHL and the significance of evaluating prognosis and directing therapy. We also inquire into whether p73 gene methylation could be used as a molecular marker for detecting minimal residual disease(MRD)in NHL. Further, inquire into whether 5-aza-2'-deoxycytiding (CdR) has the ability to recover p73 gene expression and produce antitumor effect by demethylation. The study will establish a foundation for gene therapy by inhibiting DNA methylation.Method and results: Firstly, genomic DNA is abstracted from NHL cells and denatured by hot-denaturation method before it is disposed by NaHSO₃, then MSP is used to determine whether p73 gene is methylation or not. PCR product of p73 gene methylation is 67 bp and that of unmethylation is 72 bp .The rate of p73 gene methylation in 22 NHL is 27.2%. The sample with positive strap often accompany with negative strap. The methylation rates of p73 gene CpG island in high grade malignant NHL patients is up to 42.8% while the methylation rates of p73 gene CpG island in low grade malignant NHL patients is 0%.The rates of p73 gene methylation have significant difference between patients in high grade malignant and low grade malignant.In order to analysis whether CdR has the ability to reverse methylation of p73 gene in NHL cell, we use RT-PCR to detect p73 gene expression before and after being treated with them. Firstly, abstract genomic RNA with Trizol, Synthesis cDNA by reverse transcription, then design the primer striding over the two exons, finally amplify the cDNA temple through 35 cycles. PCR product of p73 gene expression is 390 bp .Our experiment shows: NHL cell line whose CpG island is methylated can't be detected 390bp product; NHL cell line treated with CdR also shows a positive strap.Conclusions : In this study we find that the frequency of p73 gene methylation is high in NHL, which is comparable with previous reports. P73 gene is unmethylated in normal cells, so p73 gene methylation is a feature of NHL cells. The results of RT-PCT confirm the reports that CdR has demethylation action clearly, it can make p73 gene re-express. The method of p73 gene of demehtylation provides an experimental foundation for hematologic malignant clinical therapy.