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Feng Wei, Zhaoxia Wang, Yun Wu

The clinical significance of Tumor suppressor gene methylation 、expression in nodular thyroid disease. Feng Wei, Zhaoxia Wang, Yun Wu. Department of Endocrinology ,The First Affiliated Hospital of Baotou Medical College ,Inner Mongolia University of Science and Technology, China. 研究背景. 현 안.

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Feng Wei, Zhaoxia Wang, Yun Wu

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  1. The clinical significance of Tumor suppressor gene methylation 、expression in nodular thyroid disease Feng Wei, Zhaoxia Wang, Yun Wu Department of Endocrinology ,The First Affiliated Hospital of Baotou Medical College ,Inner Mongolia University of Science and Technology, China

  2. 研究背景 현 안 • Nodular thyroid diseasePathogenesis is very complex • Relationship withGenetic and environmental factors andthyroid nodular disease closely. • iodine intake abnormalities, • infections, stress • Radiation exposure. • Nodular thyroid disease pathogenesis 1.잠재 고객 Environmental factors • mutations ,Activation, tumor suppressor genes Genetic factors • Immune dysfunction, • autoantibody formation Immune factors • Thyroid cell degeneration, • inflammation Other factors It is a multi-factor, multi-gene complex processin many ways,

  3. Background DNA methylation Other factor Gene silencing The tumor suppressor gene inactivation Tumor formation DNA methylation and other factors together lead to gene silencing, causing the inactivation of tumor suppressor genes resulting in tumorigenesis

  4. Background • Detected circulating DNA in the blood of patients with malignant to genetic diagnosis in recent years has become a hot topic. • the blood circulating DNA may become a new molecular markers of tumor diagnosis 、 disease staging and prognosis effective.

  5. Background4 tumor suppressor genes DAPK--death-associated protein kinase is a tumor suppressor gene, a calcium / calmodulin-dependent serine / threonine protein kinase,Is a kind of actin filaments associated with pro-apoptotic proteins involved in the induction of apoptosis, inhibition of tumor progression and metastasis RARβ2receptor hormone nuclear receptor superfamily members, and a variety of biological effects mediated by retinoic acid, A variety of biological effects mediated by retinoic acid, plays a very important role in the regulation of cell differentiation and proliferation process RASSF1Athrough the RAS mediated signaling channel to play a variety of biological effects, and can promote apoptosis, Cell cycle arrest, and play an important role in tumorigenesis and development. PTENCaninhibit the growth of tumor cells, Promote apoptosis Involved in the regulation of the cell cycle and the inhibition of tumor metastasis The above four genes in a variety of advanced tumors including thyroid cancer, there are varying degrees of mutation or missing .

  6. Objective: • This study detected the tumor suppressor gene (PTEN, RASSF1A, DAPK, RARβ2 ) DNA methylation and mRNA expression status in the peripheral blood of patients with nodular thyroid disease. meantime,discuss its clinical significance. • For the benign and malignant thyroid nodules early identification of molecular diagnosis is based.

  7. subjects: GROUP NUMBER SEX AGENODULE SIZECALCIFICATION ECTFNACACCORD pathology M F ＞45 ＜45 ＞1cm ＜1cm YES NO COLD WARM YES NO Adenoma 50 23 27 22 28 29 21 16 34 28 22 48 2 Nodular 66 25 41 24 42 43 23 44 22 32 34 65 1 Thyroid cancer 24 13 11 10 14 16 8 17 7 19 5 21 3 GD 34 14 20 18 16 8 26 2 32 0 34 Hashimoto 40 15 25 23 17 27 13 18 12 14 2 6 40 0 control 60 27 33 30 30 Table 1 experimental groups general clinical conditions

  8. MSPExperimental methods in peripheral blood Extract the DNA template Collecting Blood samples PCR products were identified Modified DNA methylation MSP electrophoresis results Methylation specific PCR Calculated for each group gene methylation rate the rate ofEach group gene methylation Relationship between the rate of clinical data with gene methylation Conclusion

  9. RT-PCR Experimental methods Mononuclear cell separation（PBMC） Collecting Blood samples PCR products were identified Total RNA extractionCDNA was synthesized(M-MLV) RT-PCR electrophoresis Reverse transcriptase polymerase chain reaction Each group gene mRNA positive rate Gene mRNA positive expression rate Relationship between the rate of clinical data with gene mRNA expression Conclusion

  10. Result 1： Four genes MSP • The high methylation status of DAPK, RARβ2， PTEN, RASSF1A 4 genes in the peripheral blood in thyroid cancer 、adenoma 、nodular goiter groups, thyroid cancer> adenoma> nodular goiter. • RARβ2 gene hypermethylation was found in Hashimoto's thyroiditis nodules

  11. Result 1： Four genes MSP electrophoresis DAPK RARβ2 M:DM0901 50bpDNA Marker,1: Acancer patient; Sample 2: thyroid adenoma; samples: normal control group; M methylation bands, 149bp, U unmethylated bands 151bp (50bp at the primer-dimers) m :the DM0901 50bpDNA Marker; 1: cancer; 2: nodular goiter; 3: normal control group; M: methylation bands 98bp, U: non-methylation of bands, 106bp, (50bp at the primer-dimers PTEN RASSF1A

  12. AdenomaNodulesThyroid cancerGD Hashimoto Control Result 1 RASSF1A 45% 40% 35% 30% 25% 20% 15% 10% 5% 0 PTEN DAPK RARβ2 Figure 1 The methylation rate distribution of PTEN、RASSF1A、 DAPK、RARβ2 in the experimental group

  13. Result 2：Four genes mRNAexpression • DAPK, RARβ2, PTEN, RASSF1A gene mRNA expression in the peripheral blood of thyroid cancer and adenoma reduce or even missing, And was negatively correlated with 4 genes methylation. • Tip 4 genes promoter methylation may be one of the reasons causing gene expression to reduce or missing.

  14. Result 2：The four genes mRNA expression electrophoresis RARβ2 DAPK M： DM0901 100bpDNA marker; 1,2: nodular goiter; 3,4: adenoma; 5: Hashimoto's thyroiditis; 6,7: thyroid cancer; RT-PCR product of351bp; product of β-actin 550bp; M： DM0901 100bpDNA marker; 1,2: nodular goiter; 3,4: adenoma;5,6,7: thyroid cancer; RT-PCR product of 335bp; product of β-actin 550bp; RASSF1A PTEN

  15. AdenomaNodules Thyroid cancer GD Hashimoto Control Result 2： Theexpression rate distribution of PTEN、RASSF1A、 DAPK、RARβ2 mRNA in the experimental group 90% 80% 70% 60% 50% 40% 30% 20% 10% 0 RARβ2 Figure 2 RASSF1A PTEN DAPK

  16. Result 3 Clinical significance • DAPK, RARβ2 、PTEN and RASSF1A gene promoter methylation and mRNA expression are realated with lymph node metastasis in thyroid cancer patients.No relationship betweennodule size, calcification and ECT.

  17. Result 4 Clinical significance • PTEN, RASSF1A methylation and loss of expression play an important role in the elderly thyroid cancer. • PTEN, RASSFIA genes may be a prognostic indicator of thyroid cancer. • Female thyroid cancer more likely to be found in the RASSF1A gene promoter region methylation。

  18. Discussion: • Tumor suppressor genes regulate cell growth and differentiation and has the potential to inhibit tumor growth function. When they functionally inactivated or when genes deletion, mutation can lead to tumorigenesis.

  19. Discussion: • The test results found that the group of thyroid cancer and thyroid adenoma group occurred 4 geneshypermethylation and mRNA transcripts reduced or missed.

  20. Discussion: • The experiment result found a negative correlation between the methylation and mRNA expression of the 4 genes .

  21. It showed that the methylation of the 4 genes promoter region CPG can inhibit the mRNA expression ,resulting infunctional inactivationand thus participate in the incidence of thyroid cancer and thyroid adenoma.

  22. Discussion: • Be alert of the thyroid cancer when hypermethylation occured in benign thyroid adenoma. The reason maybe to discover that methylation can be detected in the peripheral blood of patients with thyroid adenoma during the tumor growth and metabolic process ,when the tissue or cellsinjure ,necroses and release DNA into the blood.

  23. Discussion: • HT is related with PTC. Some study proposes that the HT may be one of high risk factors for thyroid cancer . • The above study may suggest that RARβ2 gene promoter hypermethylation may contribute to Chronic lymphocytic thyroiditis Development of thyroid cancer.

  24. Discussions: • The methylation rate ofPTEN 、RASSF1A、 RARβ2has nothing to do with the nodule size, calcification and ECT.There is no statistically significant difference. • Methylation is related with the age、 lymph node metastasis in thyroid cancer.

  25. Conclusions: Thedetermination of MSPDNA and mRNA in peripheral blood of the pations with nodular thyroid play an important role in early diagnosis about the cancer .

  26. Thank You For Attention!

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