Methylation status of smg1 promoter in multiple myeloma patients
,1 Mehdi azad
,2,* Reza kouchaki
,3 Hamid gholipour
,4 Saeid abroun
1. Department of Medical Biotechnology, Faculty of Medicine, Babol University of Medical Sciences, babol, Iran
2. Faculty of Allied Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
3. Faculty of Allied Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
4. Department of hematology, tarbiat modares University of Medical Sciences, tehran, Iran
5. Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Multiple myeloma (mm) is the third most common hematological malignancy worldwide after leukemia and lymphoma and involves approximately 15,000 people in the world, per year. mm is characterized by neoplastic plasma cells proliferation in the bone marrow. the “myeloma cell” is a malignant plasma cell that has been released from germ line through centroblastic and centrocytic stages and produces 13,000 to 85,000 monoclonal antibodies, mainly from the igg class under influence of changes in the class of immunoglobulin, somatic hypermutation and para-protein excretion.
epigenetic modifications can be observed at any stage in the development of tumor. more than a decade ago, it has been shown that the overall level of genomic methylation in cancer cells has decreased compared to normal tissues. the hypo-methylation starts chromosome instability and oncogenesis. on the contrary, hyper-methylation leads to silencing of tumor suppressors expression.
smg1 gene (located in chr.16) that plays a central role in nmd, maintaining the telomeric integrity, protection against tnf-induced apoptosis, the lifespan regulation, embryogenesis, p53 protein activation, and dna damage response (ddr) mechanism. various studies have shown that smg1 acts as a special tumor suppressor gene in hypoxic tumors. in this study, we identified the methylation state of the smg1 promoter in mm patients.
In this study, 9 mm patients (2 females and 7 males, all were new cases) who referred to shariyati and shahid chamran hospital in tehran, and 4 healthy individuals were evaluated (confirmed by flow-cytometry). the genomic dna was extracted from peripheral blood mononuclear cells (by mini kit of geneall). the purified dna treated by sodium bisulfate (by qiagen epitect bisulfite kit). this step converts un-methylated cytosine to uracil. methylation specific pcr (msp) is performed by epitect msp qiagen kit and two specific primers (methylated specific primer and un-methylated specific primer) which approved bioinfomatically. the products have been analyzed qualitatively by agarose gel electrophoresis.
The methylation statuses of smg1 promoter evaluated as hemi-methylated in multiple myeloma patients. the presence of sharp bands indicates methylated and un-methylated dna, partially. this means the pattern of methylation of the smg1 gene in mm patients is hemi-methylated. it seems this genotype is associated with incomplete methylation of promoter regions, so it can be assumed that one pair of coupled cpg contains un-methylated c, and the other has 5hmc elements. also, the methylation statuses of smg1 promoter evaluated as hemi-methylated and un-methylated in control individual.
We found that smg1 promoter methylation statuses in mm patients are hemi-methylated compare to hemi- & un-methylated statuses in healthy individuals. so, it can suggest that smg1 have a complicated role in mm pathogenesis, different from its role as a tumor suppressor gene in other cancers. also. we suggest studying more on smg1 expression level and methylation status in the various types of cancers.
although the methylation status of smg1 cpg island has not been studied in multiple myeloma patients so far, but there are reports of the hyper-methylation status of smg1 cpg island in other cancer (such as head and neck cancer). given these findings, it looks that smg1 is thought to be a tumor suppressor gene that its expression has been reduced in some types of cancers. our study shows that smg1 expression level must be increase in mm relatively due to finding some individuals with un-methylated status in the promoter of smg1. hemi-however, more evidence is needed to prove this claim.
Methylation, smg1, methylation-specific pcr