Preliminary Studies On The Methylation Of DAZAP2 Promoter In Multiple Myeloma And Preparation Of DAZAP2 Polyclonal Antibody

Multiple myeloma(MM)is a cancer with abnormal proliferation of malignant plasma cells.To find the MM-associated gene, our study group analyzed gene expression profiles of bone marrow mononuclear cells from MM patients and control.Results showed that DAZAP2 (deleted in azoospermia associated protein 2) gene expression remarkably declined. The declined expression level of DAZAP2 was then verified by RT-PCR and Western blotting. To identify the potential mechanism of down-regulation of DAZAP2,we first analyzed its promoter region by MethPrimer. Analysis results showed that there were two CpG islands (CpG1,CpG2) in DAZAP2 promoter region, and both of them showed high-density of methylation. Methylated CpG2 might here more significant influence on the expression of DAZAP2.To illuminate the epigenetic mechanism on gene methylation, we analyzed CpG2 promoter region using TFSEARCH software. We found several transcription factor binding sites(TFBS), such as HSF, p300, CREB,ADRI etc.We selected AP-2,CREB,and c-Rel TFBS as our research targets according to the result of sulfite-genome sequencing. I labelled the probes of AP-2,CREB,and c-Rel TFBS with biotin, extract ed KM3 nuclear protein, and performed gel retardation experiment, namely electrophoretic mobility shift assay, EMSA).Results showed that the cytosine methylation of TFBS had influence on the binding of tested TF to the promoter regions, which inhibited inhibited gene expression.In order to detect DAZAP2 at protein level and establish it as a biomarker of clinical detection of MM, we tried to generate its multiclonal antiserum.DAZAP2 was first inserted into prokaryotic expression vector pQE30.We used 1 mmol/L IPTG to induce the expression of fusion protein after transformed the recombinant into E. coli JM109. The site of product was 17 KD on a SDS-PAGE gel.After purified by Ni-NTA affinity chromatography, the protein was used to immune rabbit to produce muticlonal antiserum. We detected the titer of the antibody with ELISA, and result showed the titer was 1:6400. Multiclonal antibody was prepared to detect clinical MM specimens.In conclusion, our results showed that methylated CpGs in the promoter region play a critical role in down-regulation of DAZAP2 in MM. Methylation of promoter region inhibited the binding of transcription factors and down-regulated the expression of DAZAP2. Multiclonal antibody of DAZAP2 may be of remarkable importance in clinical diagnosis of MM.