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Hypermethylation of the 16q23.1 tumor suppressor gene ADAMTS18 in clear cell renal cell carcinoma.

Xu B, Zhang L, Luo C, Qi Y, Cui Y, Ying JM, Zhang Q, Jin J - Int J Mol Sci (2015)

Bottom Line: 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.We conclude that ADAMTS18 gene hypermethylation may be involved in the tumorigenesis of ccRCC and may serve as a novel biomarker for this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, National Urological Cancer Center, 8 Xishiku Street, Xicheng District, Beijing 100034, China. xuben_pku@sina.com.

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.

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BGS high-resolution mapping of the methylation status of each CpG site (ovals) in the ADAMTS18 gene promoter region. (A) BGS results for the HEK293, A498 and Ketr-3 cell lines; and (B) BGS results for the ccRCC primary tumors (T) and adjacent normal tissues (N). The rows represent the individual alleles of the ADAMTS18 gene promoter that were analyzed. The arrows indicate MSP primer sites. The black circles indicate methylated sites, while the white circles indicate unmethylated sites.
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ijms-16-01051-f004: BGS high-resolution mapping of the methylation status of each CpG site (ovals) in the ADAMTS18 gene promoter region. (A) BGS results for the HEK293, A498 and Ketr-3 cell lines; and (B) BGS results for the ccRCC primary tumors (T) and adjacent normal tissues (N). The rows represent the individual alleles of the ADAMTS18 gene promoter that were analyzed. The arrows indicate MSP primer sites. The black circles indicate methylated sites, while the white circles indicate unmethylated sites.

Mentions: Bisulfite genomic sequencing (BGS) was performed to confirm the methylation status of the ADAMTS18 gene in the HEK293 and A498/Ketr-3 cell lines (before and after demethylation treatment) and in the three hypermethylated tumors along with their adjacent normal tissues. A typical CpG island in the ADAMTS18 gene (an 1150-bp region containing 36 CpG sites) has been amplified and identified by Jin et al. [14]. The BGS results were consistent with the MSP results, revealing a high density of hypermethylated CpG sites in the A498/Ketr-3 cell lines (before demethylation treatment) and in the hypermethylated tumor samples (Figure 4A,B). However, not every CpG site in the ADAMTS18 gene promoter region was hypermethylated, which indicated that methylation of only a subset of CpG sites could yield a positive MSP result, although unmethylated regions might be observed simultaneously.


Hypermethylation of the 16q23.1 tumor suppressor gene ADAMTS18 in clear cell renal cell carcinoma.

Xu B, Zhang L, Luo C, Qi Y, Cui Y, Ying JM, Zhang Q, Jin J - Int J Mol Sci (2015)

BGS high-resolution mapping of the methylation status of each CpG site (ovals) in the ADAMTS18 gene promoter region. (A) BGS results for the HEK293, A498 and Ketr-3 cell lines; and (B) BGS results for the ccRCC primary tumors (T) and adjacent normal tissues (N). The rows represent the individual alleles of the ADAMTS18 gene promoter that were analyzed. The arrows indicate MSP primer sites. The black circles indicate methylated sites, while the white circles indicate unmethylated sites.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307290&req=5

ijms-16-01051-f004: BGS high-resolution mapping of the methylation status of each CpG site (ovals) in the ADAMTS18 gene promoter region. (A) BGS results for the HEK293, A498 and Ketr-3 cell lines; and (B) BGS results for the ccRCC primary tumors (T) and adjacent normal tissues (N). The rows represent the individual alleles of the ADAMTS18 gene promoter that were analyzed. The arrows indicate MSP primer sites. The black circles indicate methylated sites, while the white circles indicate unmethylated sites.
Mentions: Bisulfite genomic sequencing (BGS) was performed to confirm the methylation status of the ADAMTS18 gene in the HEK293 and A498/Ketr-3 cell lines (before and after demethylation treatment) and in the three hypermethylated tumors along with their adjacent normal tissues. A typical CpG island in the ADAMTS18 gene (an 1150-bp region containing 36 CpG sites) has been amplified and identified by Jin et al. [14]. The BGS results were consistent with the MSP results, revealing a high density of hypermethylated CpG sites in the A498/Ketr-3 cell lines (before demethylation treatment) and in the hypermethylated tumor samples (Figure 4A,B). However, not every CpG site in the ADAMTS18 gene promoter region was hypermethylated, which indicated that methylation of only a subset of CpG sites could yield a positive MSP result, although unmethylated regions might be observed simultaneously.

Bottom Line: 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.We conclude that ADAMTS18 gene hypermethylation may be involved in the tumorigenesis of ccRCC and may serve as a novel biomarker for this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, National Urological Cancer Center, 8 Xishiku Street, Xicheng District, Beijing 100034, China. xuben_pku@sina.com.

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.

Show MeSH
Related in: MedlinePlus