| Multiple myeloma is a kind of incurable malignancy of end stage B-lineage cells, and characterized by an accumulation of neoplastic plasmia cells in the bone marrow and invasion into other organs. Clinical manifestations of MM because of the accumulation of plasma cells in the bone marrow and excessive levels of monoclonal immunoglobulin can result in osteolytic lesions, anema, myelosuppression and renal dysfunction. Cytogenetic, immunization and cytokine have been linked to the development fo MM. It is becoming clear that in addition to genetic aberrations, epigenetic processes play a major role in carcinogenesis. DLC-1 gene is frequently deleted in hepatocellular carcinoma. The absence or down-regulation of DLC-1 was detected in many cancer or tumor cell lines, such as, DLC-1 promoter hypermethylation has been linked to the transcription silencing of DLC and is the major epigenetic mechanism associated with loss of tumor suppressor gene function. Tannscriptional silencing promoter hypermethylation of DLC-1has been detected in over 80% of patients with acute lymphoblastic leukemia and non-Hodgkin's lymphoma. Similar consequence was also observed in MM cells lines. However, the correlation between expression and methylation of DLC-1 and clinical characteristics in MM patients has not been identified. To identify whether DLC-1 was associtated with the pathogenesis of multiple myeloma, we examined the expression and hypermethylation of DLC-1 in MM patients and MM cell line U266, observed the effect the changers of biological behaviour of U266 after treated with Decitabine at varying concentrations. In addition, to investigate the function of isform 1 of DLC-1, the eukaryotic expression vector of GFP-DLC-1 was constructed.Chapter one The study on expression and significance of DLC-1 in MMObjective The aim of the study is to examine the expression of DLC-1 in newly diagnosied MM patients, and to determine its associations with the clinical characteristics of MM, So it can provide experimental datas for MM early diagnosis, therapy, and prognosis to a certain extent.Methods RT-PCR and Real-time RT-PCR were performed to analyze the expression of DLC-1 respectly in MM patients and controls. Changes of the expression of DLC-1 in MM patients were analyzed with clinical data by correlation. Such as HB, BUN, LDH,β2-MG, IgG, IgM, KAP, LAM, ALB and Cr.Results The expression of DLC-1was measured by△Ct. MM patients has a value of 9.93±2.97, while controls was 7.55±3.0. There is a significant difference between MM patients and controls. MM patients have a decreased expression of DLC-1. But the expression of DLC-1 was no sgnificant difference between MM clinical types or stages Because the samples of patients was less and the classification or staging data distribute was skewed. The correlation among the expression of DLC-1 and clinical characteristics was analysied . A significant positve corrclation (P<0.05) was found among△Ct and Cr, the ratio of bone marrow plasm cells, while negative correlation between△Ct and Serum albumin. The decreased expression of DLC-1 is of certain significance in assessing prognosis of MM.Conclusions The expression of DLC-1 was always decreased or deleted in MM patients. A significant positve corrclation (P<0. 05) was found among△Ct and Cr, the ratio of bone marrow plasm cells, while negative correlation between△Ct and Serum albumin. The decreased expression of DLC-1 is of certain significance in assessing prognosis of MM.Chapter two The study on methylation and significance of DLC-1 in MMObjective To identify the mechanism of DLC-1 downregulation in MM, we investigated the methylation status of the DLC-1 gene. To determine the clinical significance of methylation, the relationships between the methylation status and cilical characteristics was analysied.Methods Methylation-specific PCR was used to measured the methylation status of DLC-1 in MM patients or control guoup. To study the reliability of MSP, bisulfite DNA sequencing was also carried out to examine the methylation status of CpG dinucleotide. Correlation analysis was done between the methylation status and cilical characteristics.Results The CpG island 5' to the DLC-1 gene was methylated in 12 (48%) of 25 MM patients. The methylation status was correlation with clinical stage(χ2=10.532,P=0.002), BUN(positve corrclation), Cr (χ2=8.148,P=0.008;positve corrclation)and the ratio of bone marrow plasm cells(positve corrclation). It suggested that methylation of DLC-1 played an important role in progression and prognosis assessment of MM. The results of BSP Confirmed that the status of methylation measured by MSP was reliabled.Conclusions Methylation alterations is a major mechinasim in inactivation of the DLC-1 gene in MM. The methylation status of DLC-1 played an important role in progression and prognosis assessment of MM.Chapter three The Effects of Decitabine on the Expression, methylation and Biological Behaviour of U266 cellsObjective This study was designed to explore the effects of Decitabine on the Expression, methylation and biological behaviour of U266 cells in vitro, thus to provide new ideas and experiment basement, and to provide new evidences for pathogenesis of Multiple Myoloma.Methods to determind the expression and methylation status of U266 with or without Decitabine, RT-PCR , Real-time RT-PCR, MSP and BSP were Combined. The impact of Decitabine on the ability of proliferation, migration and invasing of U266 cells was assessed by MTT test, colony formation assay,FCM analyzing cell cycle, Transwell migration test and Matrigel invasing test.Results The study demonstrated that Decitabine could significantly supress the proliferation of U266 cells in a time-dependent and a dose-dependent manner, inhibit the synthesis of DNA, block the cell cycle at G0-G1 stage, and decrease the ability of migration and invasing in low concentration. The decreased expression and CpG hypermethylation of DLC-1 can be dected in U266. Decitabine can improve the expression of DLC-1 and reverse the status of methylation.Conclusions It was concluded that Decitabine may act as a effective drug for MM by inhibiting the proliferation, migration and invasing ability, and the mechinism was correation with the improved expression and reversal methylation of DLC-1.Chapter four Construction and characterization of pEGFP-N1/DLC-1 expression vectorObjective This study was designed to construct isform 1 of DLC-1 (pEGFP-N1/DLC-1)expression vector, thus to provide experiment basement for the follow study on the location, function and Regulation pathways.Methods Total ORF of DLC-1 was amplified from pCMV-Sport 6/DLC-1 plasmid, then the amplified fragment was cloned into pMD18 T-Vector, Subsequently,the DLC-1 ORF was cloned into pEGFP-N1 vector by double restriction with Sal I and BamH I. and the eukaryotic expression vector of pEGFP-N1/DLC-1 was constructed. Recombinant vector of pEGFP-N1/DLC-1 was confirmed by PCR, dual-site endonuclease and sequencing.Results Recombinant vector of pEGFP-N1/DLC-1 was confirmed by PCR, dual-site endonuclease and sequencing. The recombinant plasmid pEGFP-N1/DLC-1 was then transfected transiently into U266 cells, The results showed that the transfected cells could display specific immunofluorscence.Conclusion Plasmid vector pEGFP-N1/DLC-1 has been successfully Constructed and it can provide experiment basement for the follow study on the location, function and Regulation pathways.
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