Study On P15 Gene Methylation And Demethylation Therapy In Multiple Myeloma

Purpose: Multiple myeloma(MM) is a kind of malignant hematologic tumors that threaten people's health and life severely. At present, the pathogenesis of MM is not understood, and its main therapy is combining chemo-therapy, assisting withinhibiting newly born blood vessel, immune therapy and hematopoietic stem cell transplantation and so on. Chemo-therapy have 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 MM, especially the role of p15 gene CpG island methylation on occurrence and development of MM and the significance of evaluating prognosis and directing therapy. We also inquire into whether p15 gene methylation could be used as a molecular marker for detecting minimal residual disease ( MRD ) in MM. Further, inquire into whether 5-aza-2'-deoxycytiding (CdR ), heplipin and arsenic trioxide have the ability to recover p15 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 MM cells and denatured by hot-denaturation method before it is disposed by NaHSO3, then MSP is used to determine whether p15 gene is methylation or not. PCR product of p15 gene methylation is 148 bp and that of unmethylation is 154 bp .The rate of p15 gene methylation in 22 MM is 63.6%. The sample with positive strap often accompany with negative strap. The tendency of p15 gene CpG island methylation rates in Complete remission(CR), initial and relapse group increases gradually, and the rate of methylation is similar before and after chemo-therapy. The rates of p15 gene methylation have no significant difference between patients in stage I , II and III. The curative efficiency of 13 cases with p15 gene methylation is 30.8%, while that of 8 cases with p15 gene unmethylation is 87.5%, there are notable difference between them(P<0.05).In order to analysis whether CdR , heplipin and arsenic trioxide have the ability to reverse methylation of p15 gene in Raji cell line, we use RT-PCR to detect p15 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 p15 gene expression is 420 bp .Our experiment shows: Raji cell line whose CpG island is methylated can't be detected 420 bp product; HL-60 cell line having unmethylated CpG island shows a positive strap; Raji cell line treated with 5X 10-6M CdR also shows a positive strap; p15 gene expression does not have detectable up-regulation after treated with heplipin; Being treated with 12.5umol/L arsenic trioxide, Raji cell line shows a positive strap.Conclusion: In this study we find that the frequency of p15 gene methylation ishigh in MM, this is comparable with previous reports. The patients with methylationhave poor responsibility to chemo-therapy and clinic remission is lower. No relationshipbetween methylation and clinic stages indicated that methylation may be an early eventin the pamogenesis of MM. P15 gene is unmethylated in nonmalignant bone marrow ornormal cells, so p15 gene methylation is a feature of MM cells. MSP for p15 gene has ahigh sensitivity, so it has the possibility to be an important marker for MRD in MM andto direct therapy. So high frequency of p15 gene methylation in MM suggests thatdemethylation may be an effective method, and that has more possibility because of thesensitivity of tumor suppressor gene inactivatied due to methylation to demethylationdrugs. Our study confirms the reports that CdR and arsenic trioxide have demethylationaction clearly, it can make p15 gene reexpress. But, in our study heplipin has nodetectable demethylation action that suggests that reverse methylation may be not itsantitumor mechanism. In our study, th...