[Frontiers in Bioscience E3, 137-157, January 1, 2011]

Aberrant methylation as a main mechanism of TSGs silencing in PTC

Karolina Czarnecka1, Dorota Pastuszak-Lewandoska1, Monika Migdalska-Sek1, Ewa Nawrot1, Jan Brzezinski2, Marek Dedecjus2, Lech Pomorski3, Ewa Brzezianska1

1Department of Molecular Bases of Medicine, Medical University of Lodz, Pomorska St. 251, 92-213 Lodz, Poland, 2Department of General, Oncological and Endocrine Surgery, Medical University of Lodz, Polish Mother's Memorial Hospital - Research Institute, Rzgowska St. 281/289, 93-338 Lodz, Poland, 3Department of General and Oncological Surgery, Medical University of Lodz, Parzeczewska St. 35, 95-100 Zgierz, Poland

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Thyroid tissue samples
3.2. Isolation of total RNA and genomic DNA
3.3. Reverse transcription reaction
3.4. Gene expression analysis: TaqMan real-time PCR assay
3.5. Promoter methylation analysis
3.5.1. Sodium bisulfite modification of DNA
3.5.2. MSP primers and methylation-specific PCR
3.5.3. BSP primers and bisulfite-specific PCR
3.5.4. Direct sequencing
3.6. Loss of heterozygosity and microsatellite instability analysis
3.6.1. Polymerase chain reaction with microsatellite markers
3.6.2. Analysis of microsatellite loci
3.7. Statistical analysis
4. Results
4.1. Results of relative expression analysis of the studied genes
4.1.1. Results of relative gene expression analysis in PTC and NG groups
4.1.2. Statistical analysis of gene expression
4.1.2.1. Comparison of RQ levels between PTC and NG groups
4.1.2.2. Comparison of RQ levels in PTC group
4.1.2.3. Comparison of RQ levels between IR and NIR genes in PTC and NG groups
4.2. Results of methylation status analysis of the studied genes
4.2.1. Gene methylation status in PTC and NG groups
4.2.2. Sequencing analysis of ARHI and CDH1 in PTC group
4.2.3. Statistical analysis of promoter methylation status
4.2.3.1. Comparison of MI values between PTC and NG groups
4.2.3.2. Comparison of MI values between IR and NIR genes in PTC and NG groups
4.2.3.3. Relationship between RQ and MI values in PTC and NG groups
4.3. Results of LOH/MSI analysis
4.3.1. Results of LOH/MSI analysis in PTC and NG groups
4.3.2. Statistical analysis of LOH/MSI frequency
4.3.2.1. Comparison of FAL index values in PTC group
4.3.2.2. Relationship between FAL index and RQ values in PTC group
4.3.2.3. Relationship between FAL index values and LOH/MSI frequency in PTC group
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

In the present study the role of tumour suppressor genes (TSGs) hypermethylation and genetic instability of LOH/MSI type in thyroid tumorigenesis was assessed. Expression, methylation status and presence of LOH/MSI were analyzed for 8 TSGs selected from imprinted (IR) and non-imprinted (NIR) chromosomal regions in papillary thyroid carcinomas (PTCs) and nodular goitres (NGs). The results show that methylation-induced gene silencing occurs at an early step of thyroid carcinogenesis and involves multiple genes. Genetic changes of LOH/MSI type are less frequent. In PTC samples, the lack of significant differences in the frequency of LOH in IR and NIR suggests that it is not a key mechanism changing the pattern of gene expression. Co-methylation observed both in NG and PTC raises a possibility that, in thyroid tissue, methylation-induced silencing may occur not only in malignant transformation but also in functional context. We did not recognize any of the studied TSGs - in regard to aberrant methylation status or LOH/MSI frequency - as a selective molecular marker in thyroid tumorigenesis.