Cis regulatory element determines CpG island methylator phenotype in urothelial carcinoma and its implication in non-invasive diagnosis using voided urine
| Autor: | Szu-Shan Chen, 陳似姍 |
|---|---|
| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 Urothelial carcinoma (UC) is the second most common urological malignancy with high rate of recurrence. Patients with UC would need to have lifetime follow-up and repeated cystoscopy for cancer detection. However the sensitivity of urine cytology, the current non-invasive diagnosis, is known to be low especially for low grade cancer patients. It’s necessary to develop novel biomarkers with high specificity and high sensitivity to improve the diagnosis of UC patients. DNA hypermethylation in CpG-rich promoters is now recognized as a common feature of human neoplasia. Interestingly, cancers with high degrees of concurrent hypermethylation (known as CpG island methylator phenotype, CIMP) present an unfavorable clinically outcome. Furthermore, CIMP-associated cancers seem to be associated with distinct histology, precursor lesions and molecular features. However, the role and molecular mechanism of CIMP in UC cancer has not been fully understood. We therefore aimed to identify hypermethylated targets that could not only classify UC patients according to their CIMP status but also serve as an accurate biomarkers for non-invasive diagnosis. In this study, using illumina 27K CpG island methylation array, we identified seven frequent hypermethylated markers (KCNG3, ZNF167, ZNF540, PRAC, NPTX2, SCNN1A, PENK) that contained similar cis-regulatory elements in their promoter in UC patient samples but not in primary normal human urothelium. First, we validated beta-value of microarray by bisulphite pyrosequencing. Further MSP analysis showed that methylation profile of these markers demonstrated a bimodal distribution, a characteristic of CIMP in UC patient samples. Interestingly, CIMP can be observed in a subset of hypermethylated targets (KCNG3, ZNF540, PRAC, SCNN1A, PENK) containing high similarity of cis-regulatory element. Bisulphite pyrosequencing and qPCR confirmed that some of these markers were epigenetically silenced by promoter methylation in bladder UC cell lines. Expression of one of the markers, PRAC, could be restored by epigenetic treatment in bladder UC cell lines. On the other hand, UC patients with higher PRAC methylation had shorter locoregional disease-free survival than those with low methylation. By using colony formation and soft agar assay, ectopic expression of PRAC in UMUC3 and J82 cell reduced cancer cell growth, anchorage independent growth, and enhanced chemosensitivity to cisplatin. We also investigated the feasibility of using DNA methylation as non-invasive biomarkers for cancer detection in urine. The sensitivity and specificity of detecting PRAC methylation in voided urine of patients using qMSP was 50% and 95%, respectively. Using a combination of methylation markers panel containing PRAC and ZNF671, the overall sensitivity for cancer detection could be increased to 75.7%. In conclusion, our result indicated that distinct DNA methylation profiles may be regulated by cell signaling through binding of transcription factors. Detection of DNA methylation in voided urine with PRAC is a sensitive and non-invasive tool for UC detection. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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