Abstract
To identify tumor suppressor genes (TSGs) silenced by hypermethylation and discover new epigenetic biomarkers for early cancer detection. ADAMTS18, located at 16q23.1, has been reported to be a critical TSG in multiple primary tumors; however, this has not yet been verified in clear cell renal cell carcinoma (ccRCC). We explored epigenetic alterations in this gene in ccRCC and analyzed possible clinicopathological associations. We examined ADAMTS18 gene expression and methylation by semi-quantitative reverse transcription PCR (RT-PCR) and methylation-specific polymerase chain reaction (MSP) in 5 ccRCC-derived cell lines before and after treatment with 5-aza-2'-deoxycytidine (5-AzaC). MSP was further performed for 101 ccRCC primary tumors and 20 adjacent normal tissues. Some cell lines and specimens were examined by subsequent bisulfite genomic sequencing (BGS) and real-time PCR. Further, we analyzed the relationship between the ADAMTS18 gene methylation and clinicopathological features, including short-term disease-free survival (DFS), in patients with ccRCC. ADAMTS18 down-regulation and hypermethylation were detected in the ccRCC-derived cell lines using RT-PCR and MSP. Treatment with 5-AzaC reversed the hypermethylation of the ADAMTS18 gene and restored its expression. Hypermethylation was further detected in 44 of 101 (43.6%) primary tumors and 3 of 20 (15.0%) adjacent normal tissues. However, a significant difference between both groups was observed (p = 0.02). BGS analysis and real-time PCR were subsequently performed to confirm the results of RT-PCR and MSP. Furthermore, the methylation status of ADAMTS18 was not significantly associated with gender, age, location, tumor diameter, pathological stage, nuclear grade or short-term DFS in patients with ccRCC (p > 0.05). The ADAMTS18 gene is often down-regulated by hypermethylation in ccRCC-derived cell lines and primary tumors, indicating its critical role as a TSG in ccRCC. We conclude that ADAMTS18 gene hypermethylation may be involved in the tumorigenesis of ccRCC and may serve as a novel biomarker for this disease.
Highlights
Clear cell renal cell carcinoma is the most common type of RCC, and it often exhibits an aggressive phenotype, including frequent metastasis to distant organs and resistance to therapeutic approaches including chemotherapy and radiotherapy [1]
To assess whether the ADAMTS18 gene was down-regulated in Clear cell renal cell carcinoma (ccRCC) tumor tissues, we initially determined its expression in ccRCC-derived cell lines using reverse transcription PCR (RT-PCR)
These results indicate that ADAMTS18 is frequently downregulated in ccRCC-derived cell lines
Summary
Clear cell renal cell carcinoma (ccRCC) is the most common type of RCC, and it often exhibits an aggressive phenotype, including frequent metastasis to distant organs and resistance to therapeutic approaches including chemotherapy and radiotherapy [1]. An increasing number of studies have demonstrated that the inactivation of tumor suppressor genes (TSGs) is a frequent event involved in the tumorigenesis of ccRCC as a result of epigenetic abnormalities in DNA methylation [2]. Previous research has demonstrated that hypermethylation of the core promoter region within CpG islands is associated with the loss of transcription of classical TSGs in multiple tumor types [3]. A large number of TSGs in a wide range of cancers have been found to be inactivated by hypermethylation of the promoter [4,5], and much of this hypermethylation occurs in the context of 5'-CpG islands, which are regions of dense accumulation of CG dinucleotides found in approximately 70% of mammalian protein-coding genes [6]. The methylation level of these genes is still low; additional TSGs with higher methylation levels in ccRCC still need to be confirmed
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