Abstract
The genome wide sequence and microarray has performed flourishingly for major cancer specimen in recent day. However, thymic epithelial tumor (TET) was rare tumor comparatively, there are few reports about epigenetic alternation in TET. Cadherin-3, also known as P-cadherin, is a protein encoded by the CDH3 gene. This study was aimed at identifying the relationship DNA methylation and gene expression of CDH3 gene in TET, especially B3 thymoma and thymic cancer. A) DNA methylation 1) DNA were extracted fresh frozen samples of B3 thymoma and thymic cancer, diagnosed as squamous cell carcinoma. These samples collected from the patients, diagnosed as TET, underwent surgery at Tokushima University from 1990 to 2016. 2) DNA was treated by bisulfite conversion. 3) DNA methylation level was measured by infinium methylation assay (Human Methylation 450K DNA Analysis Kit ; Illumina) exhaustively. 7 cases of thymic cancer and 8 cases of B3 thymoma applied this assay. 4) The methylation levels of CpG sites were calculated as β-values (β = Intensity (methylated)/intensity (methylated + unmethylated) by applying default settings of the GenomeStudio Software's DNA methylation module (Illumina). Analysis of this methylation assay used by R package. B) Immunostaining Immunostaining was performed Envision method. It was used Anti-pan-Cadherin antibody (ab16505), dilution 1300 times as first antibody. DAKO Chemomate EnVsion kit was used as second antibody. CDH3 had 2 CpG islands. 450K methylation assay set 5 CpG sites on first CpG island as promoter region. (CpG sites:B3 thymoma averageβ-values : thymic cancer averageβ-values : p-value by t-test)=(cg0900242 : 0.07±0.08 : 0.45±0.05 : 2.7×10-7), (cg07061260 : 0.21±0.21 : 0.55±0.09 : 3.3×10-3), (cg00748373 : 0.19±0.20 : 0.45±0.07 : 0.01), (cg06575065 : 0.28±0.18 : 0.50±0.07 : 0.02), (cg27417609 : 0.35±0.21 : 0.59±0.09 : 0.02). Immunostaining specimen were classified by scoring system measured by stain intensity and stain expanding. It was classified 2points; strong, 1point; weak, 0point;none, in stain intensity. It was classified 3points; diffuse (>80%), 2point;moderate(50-80%), 1point; focal (20-50%), 0point;none (<20%) in stain expanding. Expression rate (stain intensity point and stain expanding point) defined more than 4point as expression promotion. Expression promotion rate is 0% in B3 thymoma and 75% in thymic cancer. DNA methylation of CDH3 was increasing and P-cadherin protein expression was increasing in thymic cancer compared to B3 thymoma.
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