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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• Nodular thyroid disease pathogenesis
1. 잠재 고객현 안
Other factors
Immune factors
Genetic factors
Environmental factors
iodine intake abnormalities, infections, stress Radiation exposure.
mutations ,Activation,
tumor suppressor genes
Immune dysfunction,
• autoantibody formation
• Thyroid cell degeneration, • inflammation
Nodular thyroid diseasePathogenesis is very complex
Relationship with Genetic
and environmental factors
and thyroid nodular disease closely.
It is a multi-factor, multi-gene complex process in many ways,
Gene silencing
The tumor suppressor gene
inactivationTumor formation
DNA methylation Other factor
Background
DNA methylation and other factors together lead to gene silencing, causing the inactivation of tumor suppressor genes resulting in tumorigenesis
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.
Background 4 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
• RARβ2 receptor 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
• RASSF1A through 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.
• PTEN Can inhibit 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 .
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.
subjects:GROUP NUMBER SEX AGE NODULE SIZE CALCIFICATION ECT FNACACCORD
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
PCR products were identified
Extract the DNA template
Modified DNA methylation
Methylation specific PCRMSP electrophoresis
results
Calculated for each group gene methylation rate
MSP Experimental methods in peripheral blood
the rate of Each groupgene methylation
Conclusion
Relationship between the rate of clinical
data with gene methylation
Collecting
Blood
samples
PCR products were identified
Mononuclear cell separation ( PBMC )
Total RNA extractionCDNA was synthesized (M-MLV)
Reverse transcriptase polymerase
chain reaction RT-PCR electrophoresis
Each group gene mRNA positive rate
RT-PCR Experimental methods
Gene mRNA positive expression rate
Conclusion
Relationship between the rate of clinical data with gene mRNA expression
Collecting
Blood samples
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
Result 1 : Four genes MSP electrophoresis 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
RARβ2
PTEN RASSF1A
DAPK 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)
Result 1
Adenoma Nodules Thyroid cancer GD Hashimoto Control
RASSF1A
PTEN45%
40%
35%
30%
25%
20%
15%
10%
5%
0
DAPK
RARβ2
Figure 1 The methylation rate distribution of PTEN 、 RASSF1A 、DAPK 、 RARβ2 in the experimental group
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.
Result 2 : The four genes mRNA expression electrophoresis DAPK
RARβ2
PTEN RASSF1A
M : DM0901 100bpDNA marker; 1,2: nodular goiter; 3,4: adenoma;5: Hashimoto's thyroiditis; 6,7: thyroid cancer;
RT-PCR product of 351bp; 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;
Result 2 :
Adenoma Nodules Thyroid cancer GD Hashimoto Control
RASSF1A PTEN
90%
80%
70%
60%
50%
40%
30%
20%
10%
0
DAPK RARβ2 Figure 2
The expression rate distribution of PTEN 、 RASSF1A 、DAPK 、 RARβ2 mRNA in the experimental group
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 between nodule size, calcification and ECT.
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 。
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.
Discussion:
• The test results found that the group of thyroid cancer and thyroid adenoma group occurred 4 genes hypermethylation and mRNA transcripts reduced or missed.
Discussion:
The experiment result found a negative
correlation between the methylation
and mRNA expression of the 4 genes .
It showed that the methylation of the 4 genes
promoter region CPG can inhibit the mRNA
expression ,resulting in functional inactivation and thus participate in the incidence of thyroid cancer and thyroid adenoma .
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 cells injure ,necroses and release DNA into the blood.
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.
Discussions:• The methylation rate of
PTEN 、 RASSF1A 、 RARβ2 has 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.
Conclusions:
The determination of MSP DNA and mRNA in peripheral blood of the pations with nodular thyroid play an important role in early diagnosis about the cancer .
Thank You For Attention!