Huang Changjun, Tian Yuan, Peng Rui, Zhang Changhe, Wang Dong, Han Sheng, Jiao Chenyu, Wang Xing, Zhang Hai, Wang Yun, Li Xiangcheng
Department of General Surgery, Luohe Central Hospital Affiliated to Luohe Medical College, Luohe, China; Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, China.
J Gastroenterol Hepatol. 2015 Feb;30(2):421-33. doi: 10.1111/jgh.12722.
Downregulation of the WW domain containing oxidoreductase (WWOX) has been reported to be involved in tumorigenesis in several neoplasms. This study sought to investigate the expression and role of WWOX in intrahepatic cholangiocarcinoma (ICC).
WWOX expression was measured by quantitative real-time polymerase chain reaction (PCR), immunoblot, immunofluorescence, and immunohistochemistry. The prognostic significance was assessed by Kaplan-Meier and Cox regression analyses. The role of WWOX in proliferation, anchorage-independent growth, gene expression regulation, and tumorigenesis was assessed by WWOX re-expression using lentivirus. Methylation-specific PCR was performed to evaluate the methylation status of the WWOX gene regulatory region. A DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (AZA), was used to activate the endogenous WWOX gene in ICC cells both in vitro and in vivo.
The expression of WWOX in ICC tissues was much lower than that in nontumorous samples and showed reverse correlation with proliferative status. Restoration of WWOX expression resulted in suppression of the growth of WWOX-deficient ICC cells through activation of the intrinsic apoptotic signaling pathway, but did not affect growth of WWOX-sufficient human intrahepatic biliary epithelial derived non-cancer cells. Multivariate analyses revealed that downregulation of WWOX was an unfavorable predictor for overall survival and cumulative recurrence rates. The WWOX gene regulatory region was frequently methylated in ICC tissues and cell lines, and intratumoral WWOX restoration, through AZA injection, suppressed tumor growth in nude mice.
Downregulation of WWOX may occur as a result of hypermethylation and implies a poor prognosis in ICC; WWOX re-expression may be a potential molecular therapeutic target for ICC.
据报道，含WW结构域的氧化还原酶（WWOX）下调与多种肿瘤的发生有关。本研究旨在探讨WWOX在肝内胆管癌（ICC）中的表达及作用。
采用定量实时聚合酶链反应（PCR）、免疫印迹、免疫荧光和免疫组化检测WWOX表达。通过Kaplan-Meier和Cox回归分析评估预后意义。利用慢病毒重新表达WWOX，评估其在增殖、非锚定依赖性生长、基因表达调控和肿瘤发生中的作用。进行甲基化特异性PCR评估WWOX基因调控区的甲基化状态。使用DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（AZA）在体外和体内激活ICC细胞中的内源性WWOX基因。
ICC组织中WWOX的表达远低于非肿瘤样本，且与增殖状态呈负相关。WWOX表达的恢复通过激活内源性凋亡信号通路抑制了缺乏WWOX的ICC细胞的生长，但不影响富含WWOX的人肝内胆管上皮来源的非癌细胞的生长。多变量分析显示，WWOX下调是总生存和累积复发率的不良预测指标。WWOX基因调控区在ICC组织和细胞系中频繁甲基化，通过注射AZA实现瘤内WWOX恢复可抑制裸鼠肿瘤生长。
WWOX下调可能是高甲基化的结果，提示ICC预后不良；WWOX重新表达可能是ICC潜在的分子治疗靶点。
