Tezval Hossein, Dubrowinskaja Natalia, Peters Inga, Reese Christel, Serth Katrin, Atschekzei Faranaz, Hennenlotter Jörg, Stenzl Arnulf, Kuczyk Markus A, Serth Jürgen
Department of Urology and Urological Oncology, Hannover Medical School, Hannover, Germany.
Department of Molecular Biology, Hannover Medical School, Hannover, Germany.
PLoS One. 2016 Oct 3;11(10):e0163873. doi: 10.1371/journal.pone.0163873. eCollection 2016.
The relevance of Corticotropin Releasing Hormone (CRH)-system in human malignancies is a question of growing interest. Here we investigated hypermethylation and epigenetic silencing of the CRH-Binding Protein (CRHBP) gene in clear cell renal cell cancer (ccRCC). Relative methylation of the CRHBP CpG island (CGI) was determined in 17 tumor cell lines as well as 86 ccRCC samples and 66 paired normal tissues using pyrosequencing and quantitative methylation specific PCR of bisulfite converted DNA. Results were statistically compared with relative mRNA expression levels of CRHBP and clinicopathological parameters of patients. Re-expression of CRHBP following 5-aza-2´-deoxycytidine treatment was investigated by quantitative mRNA expression analysis. Real-time impedance analysis was applied for analysis of invasiveness of renal tumor cells following si-RNA knockdown of CRHBP expression or ectopic expression of CRHBP. We found the CRHBP CGI to be frequently methylated in tumor cell lines of renal, prostatic, and bladder cancer. Comparison of methylation in normal and paired renal cancer tissue specimens revealed hypermethylation of the CRHBP CGI in tumors (p<110-12). DNA methylation and decreased mRNA expression were correlated (R = 0.83, p<110-12). Tumor cell lines showed 5-aza-2´-deoxycytidine dependent reduction of methylation and re-expression of CRHBP was associated with altered cellular invasiveness of renal cancer cells in real-time impedance invasion assays. Hypermethylation and inverse relationship with mRNA expression were validated in silico using the TCGA network data. We describe for the first time tumor specific epigenetic silencing of CRHBP and statistical association with aggressive tumors thus suggesting the CRH system to contribute to the development of kidney cancer.
促肾上腺皮质激素释放激素（CRH）系统在人类恶性肿瘤中的相关性是一个越来越受关注的问题。在此，我们研究了透明细胞肾细胞癌（ccRCC）中CRH结合蛋白（CRHBP）基因的高甲基化和表观遗传沉默。使用焦磷酸测序和亚硫酸氢盐转化DNA的定量甲基化特异性PCR，在17种肿瘤细胞系以及86个ccRCC样本和66个配对的正常组织中测定CRHBP CpG岛（CGI）的相对甲基化。将结果与CRHBP的相对mRNA表达水平和患者的临床病理参数进行统计学比较。通过定量mRNA表达分析研究5-氮杂-2'-脱氧胞苷处理后CRHBP的重新表达。应用实时阻抗分析来分析CRHBP表达的si-RNA敲低或CRHBP的异位表达后肾肿瘤细胞的侵袭性。我们发现CRHBP CGI在肾、前列腺和膀胱癌的肿瘤细胞系中经常发生甲基化。正常和配对肾癌组织标本中甲基化的比较显示肿瘤中CRHBP CGI的高甲基化（p<1×10-12）。DNA甲基化与mRNA表达降低相关（R = 0.83，p<1×10-12）。肿瘤细胞系显示5-氮杂-2'-脱氧胞苷依赖性甲基化降低，并且在实时阻抗侵袭试验中，CRHBP的重新表达与肾癌细胞的细胞侵袭性改变相关。使用TCGA网络数据在计算机上验证了高甲基化和与mRNA表达的反比关系。我们首次描述了CRHBP的肿瘤特异性表观遗传沉默以及与侵袭性肿瘤的统计学关联，因此表明CRH系统有助于肾癌的发展。
