Xu Ben, Zhang Lian, Luo Cheng, Qi Yan, Cui Yun, Ying Jian-Ming, Zhang Qian, Jin Jie
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.
Department of Urology, Affiliated Hospital of Taishan Medical University, Tai'an 271000, China.
Int J Mol Sci. 2015 Jan 5;16(1):1051-65. doi: 10.3390/ijms16011051.
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.
为了鉴定因高甲基化而沉默的肿瘤抑制基因（TSG），并发现用于早期癌症检测的新的表观遗传生物标志物。ADAMTS18位于16q23.1，据报道在多种原发性肿瘤中是一种关键的TSG；然而，这尚未在透明细胞肾细胞癌（ccRCC）中得到验证。我们探究了ccRCC中该基因的表观遗传改变，并分析了可能的临床病理相关性。我们通过半定量逆转录PCR（RT-PCR）和甲基化特异性聚合酶链反应（MSP）检测了5种ccRCC来源的细胞系在用5-氮杂-2'-脱氧胞苷（5-AzaC）处理前后的ADAMTS18基因表达和甲基化情况。对101例ccRCC原发性肿瘤和20例相邻正常组织进一步进行了MSP检测。部分细胞系和标本随后通过亚硫酸氢盐基因组测序（BGS）和实时PCR进行检测。此外，我们分析了ccRCC患者中ADAMTS18基因甲基化与临床病理特征之间的关系，包括短期无病生存期（DFS）。使用RT-PCR和MSP在ccRCC来源的细胞系中检测到ADAMTS18下调和高甲基化。用5-AzaC处理可逆转ADAMTS18基因的高甲基化并恢复其表达。在101例原发性肿瘤中的44例（43.6%）和20例相邻正常组织中的3例（15.0%）进一步检测到高甲基化。然而，两组之间观察到显著差异（p = 0.02）。随后进行BGS分析和实时PCR以确认RT-PCR和MSP的结果。此外，ADAMTS18的甲基化状态与ccRCC患者的性别、年龄、位置、肿瘤直径、病理分期、核分级或短期DFS均无显著相关性（p > 0.05）。ADAMTS18基因在ccRCC来源的细胞系和原发性肿瘤中常因高甲基化而下调，表明其在ccRCC中作为TSG的关键作用。我们得出结论，ADAMTS18基因高甲基化可能参与ccRCC的肿瘤发生，并可能作为该疾病的一种新型生物标志物。
