Promoter methylation quantification of four tumor suppressor gene in papillary thyroid cancer tissues
,2 Bagher larijani
,3 Ramin heshmat
,4 Mahsa mohammadamoli
,5 Seyed mohammad tavangar
1. Chronic Diseases Research Center, Endocrinology and Metabolism Population Sciences Institute,TUMS
2. Molecular Medicine Departments, Pasteur Institute of Iran, Tehran, Iran
3. Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, TUMS
4. Chronic Diseases Research Center, Endocrinology and Metabolism Population Sciences Institute, TUMS
5. Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, TUMS
6. Departments of Pathology, Dr. Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
Endocrine tumors are endocrine system related malignancies like thyroid that is the most common type with mounting trends over the last three decades all over the world. papillary thyroid cancer (ptc) is making four fifths of all thyroid cancers. finding some detection markers in order to discriminate malignant from benign one before metastasis could be really important for thyroid cancer patients and clinicians. epigenetic silencing through aberrant dna methylation of tumor suppressor genes linked to a variety of devastating consequences of human cancer. we determined the quantity of methylation in twelve candidate promoter regions of four tumor suppressor genes using the methylation-sensitive high resolution melting (ms-hrm) assay.
Fresh frozen tissues of 57 ptc patients and 45 goiter patients were collected after surgery. dna was extracted using the dneasy blood & tissue kit according to the manufacturer’s protocol. dna purity and quantity was determined using a thermo scientific™ nanodrop™ spectrophotometers 2000c spectrophotometer and then stored at -80°c. for bisulfite treatment dna from each sample were treated with sodium bisulfite conversion kit. the ms- hrm analyses were run based on the three main steps of holding, cycling, and melt curve. statistical analysis were done by statistical package for science software (spss) version 16.0. and p <0.05 was measured statistically significant.
Results: promoter methylation of four slc5a8, rassf1, mgmt and dnmt1 genes has meaningful differences between ptc cases and goiter controls.
promoter region methylation* ptc cases
number (percent) goiter cases
number (percent) p-value
slc5a8 u 17(29.8%) 35(77.8%) 0.002
m 40 (70.2%) 10 (22.2%)
rassf1 u 13(22.8%) 38 (84.4%) <0.001
m 40 (70.2%) 10 (22.2%)
mgmt u 8 (14.0%) 35 (77.8%) 0.001
m 49 (86.0%) 10 (22.10%)
dnmt1 u 29 (51.8%) 32 (74.4%) 0.018
m 27 (48.2%) 11 (25.6%)
Conclusion: among the different selected promoter region of four tumor suppressor genes the slc5a8 (b), slc5a8 (c), rassf1 (a), rassf1 (b) , mgmt (a), mgmt (c), mgmt (d), and dnmt1 (b) methylation status were meaningfully different between ptc cases and controls. in spite of the fact that dnmt1 is a denovo methyl trasferase enzyme its promoter hypermethylation was not as significant as slc5a8, rassf1, and mgmt.
acknowledgments: this article was a part of a larger project which was granted by the national institute for medical research development (nimad, grant number: 957222).
Ptc, ms-hrm, slc5a8, rassf1, mgmt, dnmt1