Internal Medicine D, Nephrology, Hypertension and Rheumatology, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany.
Institute of Sports Medicine, Molecular Genetics of Cardiovascular Disease, University Hospital Muenster, Horstmarer Landweg 39, 48149 Muenster, Germany.
Clin Epigenetics. 2017 Oct 6;9:109. doi: 10.1186/s13148-017-0415-6. eCollection 2017.
KIBRA has been suggested as a key regulator of the Hippo signaling pathway, regulating organ size, cell contact inhibition, tissue regeneration as well as tumorigenesis and cystogenesis. We recently reported that human expression depends on a complex alternative CpG-rich promoter system. Our current study aimed at the identification of epigenetic mechanisms associated with alterations in expression regulation.
We identified two separated methylation-sensitive CpG islands located to independent promoter regions. In vitro promoter methylation analysis using human neuroblastoma (SH-SY5Y) and immortalized kidney cells (IHKE) revealed that total promoter methylation by CpG methyltransferase I resulted in complete abrogation of transcriptional activity ( < 0.001), while partial methylation by II selectively repressed core promoter activity in kidney cells ( < 0.001). Cell culture-based experiments demonstrated that 5-azacitidine may be used to restore KIBRA mRNA and protein levels, while overexpression of transcription factor SP1 also induced upregulation (all < 0.001). Furthermore, SP1 transactivation of transcription was largely prevented by methylation of regulatory elements ( < 0.001). Analysis of human kidney biopsies revealed that promoter methylation was associated with human clear cell renal cell carcinoma (ccRCC; = 8 vs 16 controls, OR = 1.921, [CI 95% = 1.369-2.695]). The subsequent determination of KIBRA mRNA levels by real-time PCR in a larger patient sample confirmed significantly reduced KIBRA expression in ccRCC ( = 32) compared to non-neoplastic human kidney tissue samples (controls, = 32, < 0.001).
We conclude that epigenetic downregulation of tumor suppressor KIBRA may involve impaired SP1 binding to functional methylation-sensitive promoter elements as observed in human kidney clear cell carcinoma. Our findings provide a pathophysiological basis for future studies on altered regulation in clinical disease entities such as renal cancer.
KIBRA 被认为是 Hippo 信号通路的关键调节因子，调节器官大小、细胞接触抑制、组织再生以及肿瘤发生和囊形成。我们最近报道，人类 表达依赖于一个复杂的、富含 CpG 的替代启动子系统。我们目前的研究旨在确定与 表达调控改变相关的表观遗传机制。
我们鉴定出两个位于独立 启动子区域的分离的甲基化敏感 CpG 岛。使用人神经母细胞瘤（SH-SY5Y）和永生化肾细胞（IHKE）进行的体外启动子甲基化分析表明，CpG 甲基转移酶 I 导致的总启动子甲基化完全阻断了转录活性（<0.001），而 II 选择性地抑制了肾细胞中 核心启动子活性（<0.001）。基于细胞培养的实验表明，5-氮杂胞苷可用于恢复 KIBRA mRNA 和蛋白水平，而转录因子 SP1 的过表达也诱导了 上调（均<0.001）。此外， 调节元件的甲基化极大地阻止了 SP1 对 转录的激活（<0.001）。对人肾活检的分析表明， 启动子甲基化与人类透明细胞肾细胞癌（ccRCC；=8 与 16 个对照，OR=1.921，[95%CI 1.369-2.695]）相关。在更大的患者样本中通过实时 PCR 对 KIBRA mRNA 水平的后续测定证实，与非肿瘤性人肾组织样本（对照，=32，<0.001）相比，ccRCC 中 KIBRA 表达显著降低。
我们的结论是，肿瘤抑制因子 KIBRA 的表观遗传下调可能涉及 SP1 结合到功能性甲基化敏感的 启动子元件的能力受损，如在人类肾透明细胞癌中观察到的那样。我们的发现为未来在肾肿瘤等临床疾病实体中改变 调节的研究提供了病理生理学基础。
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