Epigenetic inactivation of transforming growth factor-β1 target gene HEYL, a novel tumor suppressor, is involved in the P53-induced apoptotic pathway in hepatocellular carcinoma
Epigenetic inactivation of transforming growth factor-β1 target gene HEYL, a novel tumor suppressor, is involved in the P53-induced apoptotic pathway in hepatocellular carcinoma
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DOI：
10.1111/hepr.12414
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发表时间：
2015-07-01
影响因子：
4.2
通讯作者：
Cheng，Kuang-Hung
中科院分区：
文献类型：
--
作者：
Kuo，Kung-Kai;Jian，Shu-Fang;Cheng，Kuang-Hung
AimHairy/enhancer‐of‐split related with YRPW motif‐like（HEYL）蛋白是最早发现的一种转录抑制因子。它是Notch和转化生长因子-β途径的下游基因。关于其在肝细胞癌（HCC）发病机制中的作用知之甚少。方法通过逆转录定量聚合酶链反应（RT-qPCR）和免疫组化（IHC）分析80例手术切除的配对HCC和邻近非癌组织的HEYL表达。用pHEYL-EGFP载体转染HCC细胞以过表达HEYL基因或用特异性shHEYL慢病毒载体感染以沉默HEYL基因表达。HEYL的表达分析和功能特性进行了评估3-（4 5-二甲基噻唑-2-基）-2 5-二苯基四唑溴化物测定，流式细胞术，RT-qPCR，免疫印迹和甲基化特异性PCR.ResultsWe确定，HEYL表达失活超过75%的HCC。此外，HEYL在SK‐Hep 1细胞中的过表达通过半胱天冬酶3和聚（ADP‐核糖）聚合酶的切割引起细胞凋亡。我们发现HEYL凋亡之前是丝氨酸15磷酸化和P53的积累。分子分析显示HEYL过表达导致p16、p19、p21、p27和Bad蛋白表达增加，而c-Myc、Bcl-2和Cyclin B1表达减少。HEYL表达的表观遗传沉默的DNA超甲基化在HCC中直接相关的HEYL表达的损失hCC.ConclusionHEYL是经常下调启动子甲基化在HCC中。HEYL可能通过上调P53基因表达和激活P53介导的凋亡而成为肝癌发生的肿瘤抑制剂。
AimHairy/enhancer‐of‐split related with YRPW motif‐like (HEYL) protein was first identified as a transcriptional repressor. It is a downstream gene of the Notch and transforming growth factor‐β pathways. Little is known about its role in the pathogenesis of hepatocellular carcinoma (HCC).MethodsEighty surgically resected paired HCC and adjacent non‐cancerous tissues were analyzed for HEYL expression by reverse transcription quantitative polymerase chain reaction (RT–qPCR) and immunohistochemistry (IHC). HCC cells were transfected with pHEYL‐EGFP vector to overexpress the HEYL gene or infected with specific shHEYL lentiviral vector to silence HEYL gene expression. HEYL expressional analysis and functional characterization were assessed by 3‐(4 5‐dimethylthiazol‐2‐yl)‐2 5‐diphenyltetrazolium bromide assays, flow cytometry, RT–qPCR, western blotting and methylation‐specific PCR.ResultsWe determined that HEYL expression was inactivated in more than 75% of HCC. In addition, overexpression of HEYL in SK‐Hep 1 cells caused apoptosis by the cleavage of caspase 3 and poly (ADP‐ribose) polymerase. We discovered that HEYL apoptosis was preceded by serine 15 phosphorylation and accumulation of P53. Molecular analysis revealed that HEYL overexpression led to increased p16, p19, p21, p27 and Bad protein expression, and reduced c‐Myc, Bcl‐2 and Cyclin B1 expression. Epigenetic silencing of HEYL expression by DNA hypermethylation in HCC directly correlated with loss of HEYL expression in HCC.ConclusionHEYL is frequently downregulated by promoter methylation in HCC. HEYL may be a tumor suppressor of liver carcinogenesis through upregulation of P53 gene expression and activation of P53‐mediated apoptosis.