候选抑癌基因MAPK10在原发性肝细胞肝癌中表达的表观遗传学调控机制及临床意义研究
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摘要
原发性肝细胞肝癌（Hepatocellular carcinoma, HCC）是人类最常见、最凶险的恶性肿瘤之一，其发生发展涉及到遗传学、表观遗传学调控及多基因改变的级联过程，最终导致癌基因的激活或抑癌基因的失活。近年来研究表明，通过表观遗传学调控机制导致的抑癌基因功能丢失与各种肿瘤包括HCC的发生发展密切相关，抑癌基因甲基化有望作为一种特异性的分子标志物在肿瘤的早期诊断、预后评估中发挥重要作用。发现更多新的有价值的抑癌基因、建立完善的HCC甲基化谱，对HCC的早期诊断、预测转移复发、提示预后和个性化治疗均具有重要的价值。
MAPK10又名JNK3，从属于促分裂素原活化蛋白激酶（mitogen-activated protein kinases, MAPK）大家族中的JUN氨基末端激酶(Jun N-terminal kinase, JNK)亚群，最近的研究提示其可能为一候选抑癌基因。在本文中，我们以MAPK10为研究目标，通过其启动子区甲基化水平与该基因在HCC细胞表达的关系来揭示MAPK10在HCC中表达的表观遗传学调控机制，同时通过载体的构建和转染、亚细胞定位、克隆的筛选和计数、流式细胞仪及TUNEL法对细胞凋亡的检测、凋亡相关基因表达的观察、联合常规化疗药物对HCC细胞毒作用的分析等方法来明确该基因对人HCC细胞的作用，并进一步在临床标本中探讨MAPK10表达沉默的临床意义。结果表明：MAPK10基因在几乎所有的正常组织包括肝组织中表达，但在HCC细胞株中却有67%被沉默或表达显著降低（8/12），且与相应的启动子区甲基化状态具有良好的相关性（58%，7/12)。从HCC的石蜡标本中也观察到，MAPK10在HCC组织中往往表达阴性或弱阳性，较癌旁组织显著降低（63%,29/46; p=0.000, Wilcoxon signed rank test）。我们进一步检测了临床收集的18例HCC标本的MAPK10基因启动子甲基化水平，发现12例存在启动子甲基化（67%）。在这18例HCC冰冻标本中同时进行了免疫组化染色检测MAPK10表达（其中15例染色成功），观察到其中11例标本的MAPK10表达缺失，而相应的这些标本的甲基化状态检测结果恰好均为阳性。为了进一步证实MAPK10基因沉默与启动子甲基化之间的关系，我们利用去甲基化药物5-氮杂-2-脱氧胞苷（5-aza-2’-deoxycytidine，5-Aza-dC）联合组蛋白去乙酰化酶抑制剂曲古菌素A（Trichostatin A，TSA）作用于MAPK10沉默的HCC细胞株HepG2和Hep3B，结果提示：伴随MAPK10基因甲基化状态的改变，其在HCC细胞中的表达也得以恢复，说明MAPK10在HCC细胞中的丢失与其启动子高甲基化密切相关。细胞功能试验则表明，通过载体构建、基因转染后，MAPK10可显著抑制HCC细胞的克隆形成、诱导凋亡发生、并能增强HCC细胞对化疗药物5-氟尿嘧啶（5-Fluorouracil,5-Fu）的敏感性。更有意义的是，我们观察到MAPK10转入HCC细胞后，凋亡相关基因p53及Caspase家族的mRNA表达及蛋白表达均较前明显增强。最后，我们研究了59例HCC病人中MAPK10表达与临床特点、预后的关系。结果表明，MAPK10表达缺失与HCC进展分期、肝内转移灶发生、高水平甲胎蛋白（AFP）显著相关；MAPK10沉默的HCC患者较MAPK10表达的患者预后更差，生存时间明显缩短。
总之，我们的研究表明，MAPK10是HCC发生发展中的一个重要抑癌基因，表观遗传学-DNA甲基化机制是其表达的调控机制之一；MAPK10在HCC中的表达与HCC患者的临床分期及预后密切相关，其表达丢失往往提示HCC病人预后更差。针对MAPK10在HCC的甲基化研究将为HCC的早期诊断、临床治疗提供有价值的新靶点。
Hepatocellular carcinoma (HCC) is one of the most lethalmalignancies for humans. Given that patients with HCC are alwaysdiagnosed at the advanced stage and HCC is one of the well identifiedcancers that are most resistant to systemic chemotherapy, efficacy ofcurative surgery or chemotherapy on HCC remains very poor, whichhighlights the need for reliable biomarkers in the early diagnosis of HCC.As for many other tumors, development of HCC is the consequence of amultistep process with genetic and epigenetic alterations in regulatorygenes, resulting in activation of oncogenes and inactivation of TSG.Promoter CpG methylation, one of the epigenetic alterations which occurfrequently during tumor development and progression, causes the loss ofTSG functions. Recent studies have evidenced the importance of promoterCpG methylation in initiating carcinogenesis and it has been hypothesizedthat TSG methylation can be used as epigenetic biomarkers for tumor diagnosis or prognosis prediction clinically. To find more valuable TSGsfrequently methylated in HCC and establish a specific methylation profilewould be helpful to provide new and reliable biomarkers for HCCmanagement.
Mitogen-Activated Protein Kinase10(MAPK10) is a member of thec-Jun N-terminal kinases (JNK) subgroup in MAP kinase superfamily,recently suggested as a tumor suppressor inactivated epigenetically. Here,we investigated its role as a tumor suppressor in HCC from the epigeneticway. MAPK10was expressed in almost all normal tissues including liver,however, it was low or silenced in67%(8/12) of HCC cell lines. For thoseclinical samples, MAPK10expression was also significantly lower in HCCthan that in adjacent non-tumor tissues (63%,29/46; p=0.000, Wilcoxonsigned rank test). Promoter methylation of MAPK10was further detectedin58%(7/12) of the HCC cell lines and in66%(12/18) of primary HCCtissues by methylation specific PCR (MSP), which was well correlated withits silenced or downregulated expression. Moreover, the transcriptionalsilencing of MAPK10could be reversed by pharmacologic demethylationand ectopic expression of MAPK10in silenced HCC cell lines couldsignificantly inhibit colony formation ability, induce apoptosis or enhancethe chemosensitivities of HCC cells to5-Fu. In addition, the expression ofp53, caspase3and caspase8could be increased after the ectopicexpression of MAPK10in these silenced HCC cells. Finally, the clinical significance of MAPK10in a cohort of59HCC cases was assessed. Inthese cases examined, negative expression of MAPK10was significantlyassociated with advanced tumour stage (p=0.001), more microsatellitenodules (p=0.025), higher serum AFP (p=0.05) and shorter overall survivaltime of HCC patients (p=0.008).
Thus, MAPK10appears to be a functional tumor suppressor gene (TSG)frequently methylated in HCC, which could be a valuable biomarker or anew therapy target clinically.
MAPK10又名JNK3，从属于促分裂素原活化蛋白激酶（mitogen-activated protein kinases, MAPK）大家族中的JUN氨基末端激酶(Jun N-terminal kinase, JNK)亚群，最近的研究提示其可能为一候选抑癌基因。在本文中，我们以MAPK10为研究目标，通过其启动子区甲基化水平与该基因在HCC细胞表达的关系来揭示MAPK10在HCC中表达的表观遗传学调控机制，同时通过载体的构建和转染、亚细胞定位、克隆的筛选和计数、流式细胞仪及TUNEL法对细胞凋亡的检测、凋亡相关基因表达的观察、联合常规化疗药物对HCC细胞毒作用的分析等方法来明确该基因对人HCC细胞的作用，并进一步在临床标本中探讨MAPK10表达沉默的临床意义。结果表明：MAPK10基因在几乎所有的正常组织包括肝组织中表达，但在HCC细胞株中却有67%被沉默或表达显著降低（8/12），且与相应的启动子区甲基化状态具有良好的相关性（58%，7/12)。从HCC的石蜡标本中也观察到，MAPK10在HCC组织中往往表达阴性或弱阳性，较癌旁组织显著降低（63%,29/46; p=0.000, Wilcoxon signed rank test）。我们进一步检测了临床收集的18例HCC标本的MAPK10基因启动子甲基化水平，发现12例存在启动子甲基化（67%）。在这18例HCC冰冻标本中同时进行了免疫组化染色检测MAPK10表达（其中15例染色成功），观察到其中11例标本的MAPK10表达缺失，而相应的这些标本的甲基化状态检测结果恰好均为阳性。为了进一步证实MAPK10基因沉默与启动子甲基化之间的关系，我们利用去甲基化药物5-氮杂-2-脱氧胞苷（5-aza-2’-deoxycytidine，5-Aza-dC）联合组蛋白去乙酰化酶抑制剂曲古菌素A（Trichostatin A，TSA）作用于MAPK10沉默的HCC细胞株HepG2和Hep3B，结果提示：伴随MAPK10基因甲基化状态的改变，其在HCC细胞中的表达也得以恢复，说明MAPK10在HCC细胞中的丢失与其启动子高甲基化密切相关。细胞功能试验则表明，通过载体构建、基因转染后，MAPK10可显著抑制HCC细胞的克隆形成、诱导凋亡发生、并能增强HCC细胞对化疗药物5-氟尿嘧啶（5-Fluorouracil,5-Fu）的敏感性。更有意义的是，我们观察到MAPK10转入HCC细胞后，凋亡相关基因p53及Caspase家族的mRNA表达及蛋白表达均较前明显增强。最后，我们研究了59例HCC病人中MAPK10表达与临床特点、预后的关系。结果表明，MAPK10表达缺失与HCC进展分期、肝内转移灶发生、高水平甲胎蛋白（AFP）显著相关；MAPK10沉默的HCC患者较MAPK10表达的患者预后更差，生存时间明显缩短。
总之，我们的研究表明，MAPK10是HCC发生发展中的一个重要抑癌基因，表观遗传学-DNA甲基化机制是其表达的调控机制之一；MAPK10在HCC中的表达与HCC患者的临床分期及预后密切相关，其表达丢失往往提示HCC病人预后更差。针对MAPK10在HCC的甲基化研究将为HCC的早期诊断、临床治疗提供有价值的新靶点。
Hepatocellular carcinoma (HCC) is one of the most lethalmalignancies for humans. Given that patients with HCC are alwaysdiagnosed at the advanced stage and HCC is one of the well identifiedcancers that are most resistant to systemic chemotherapy, efficacy ofcurative surgery or chemotherapy on HCC remains very poor, whichhighlights the need for reliable biomarkers in the early diagnosis of HCC.As for many other tumors, development of HCC is the consequence of amultistep process with genetic and epigenetic alterations in regulatorygenes, resulting in activation of oncogenes and inactivation of TSG.Promoter CpG methylation, one of the epigenetic alterations which occurfrequently during tumor development and progression, causes the loss ofTSG functions. Recent studies have evidenced the importance of promoterCpG methylation in initiating carcinogenesis and it has been hypothesizedthat TSG methylation can be used as epigenetic biomarkers for tumor diagnosis or prognosis prediction clinically. To find more valuable TSGsfrequently methylated in HCC and establish a specific methylation profilewould be helpful to provide new and reliable biomarkers for HCCmanagement.
Mitogen-Activated Protein Kinase10(MAPK10) is a member of thec-Jun N-terminal kinases (JNK) subgroup in MAP kinase superfamily,recently suggested as a tumor suppressor inactivated epigenetically. Here,we investigated its role as a tumor suppressor in HCC from the epigeneticway. MAPK10was expressed in almost all normal tissues including liver,however, it was low or silenced in67%(8/12) of HCC cell lines. For thoseclinical samples, MAPK10expression was also significantly lower in HCCthan that in adjacent non-tumor tissues (63%,29/46; p=0.000, Wilcoxonsigned rank test). Promoter methylation of MAPK10was further detectedin58%(7/12) of the HCC cell lines and in66%(12/18) of primary HCCtissues by methylation specific PCR (MSP), which was well correlated withits silenced or downregulated expression. Moreover, the transcriptionalsilencing of MAPK10could be reversed by pharmacologic demethylationand ectopic expression of MAPK10in silenced HCC cell lines couldsignificantly inhibit colony formation ability, induce apoptosis or enhancethe chemosensitivities of HCC cells to5-Fu. In addition, the expression ofp53, caspase3and caspase8could be increased after the ectopicexpression of MAPK10in these silenced HCC cells. Finally, the clinical significance of MAPK10in a cohort of59HCC cases was assessed. Inthese cases examined, negative expression of MAPK10was significantlyassociated with advanced tumour stage (p=0.001), more microsatellitenodules (p=0.025), higher serum AFP (p=0.05) and shorter overall survivaltime of HCC patients (p=0.008).
Thus, MAPK10appears to be a functional tumor suppressor gene (TSG)frequently methylated in HCC, which could be a valuable biomarker or anew therapy target clinically.
引文
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