组蛋白甲基化转移酶SETD...鼻咽癌细胞增殖和迁移的影响_封慕茵.pdf

中国病理生理杂志 Chinese Journal of Pathophysiology 2023，39（2）：259-268杂志网址：http:/组蛋白甲基化转移酶SETD4对鼻咽癌细胞增殖和迁移的影响*封慕茵1，郑阿秀1，白建荣1，曾玉梅4，罗泊涛1，申志华1，揭伟1，2，3（1广东医科大学基础医学院病理学系，广东 湛江 524023；2海南医学院第一附属医院肿瘤内科，海南 海口 570102；3海南医学院肿瘤研究所，海南 海口 571199；4中山市人民医院病理科，广东 中山 528400）摘要 目的：明确组蛋白甲基化转移酶含SET结构域蛋白4（SET-domain-containing protein 4，SETD4）在临床鼻咽癌（nasopharyngeal carcinoma，NPC）组织中的表达，分析SETD4对NPC细胞增殖和迁移的影响。方法：采用免疫组织化学法检测86例临床NPC标本与对照组30例鼻咽黏膜慢性炎症（nasopharyngeal chronic inflammation，NPI）组织中SETD4蛋白的表达；基于CRISPR/Cas9技术敲除CNE2细胞中SETD4基因，DNA测序结合半定量RT-PCR进行鉴定；以SETD4敲除前后的CNE2细胞为研究对象，于倒置显微镜下观察细胞形态，采用CCK-8实验分析细胞增殖活性、Transwell实验观察细胞迁移能力变化、Western blot检测增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）、细胞周期相关蛋白、上皮-间充质转化标志物和组蛋白赖氨酸甲基化的变化，并通过生物信息学富集SETD4相关联的功能和信号通路。结果：SETD4蛋白主要定位于NPC细胞核中，NPC组织中SETD4阳性表达率显著低于NPI对照组（P0.01）。基于CRISPR/Cas9技术成功敲除了CNE2细胞的SETD4基因，获得基因敲除的纯合子细胞。与野生型（wild-type，WT）细胞株相比，SETD4敲除（knockout，KO）细胞株呈现更明显的梭形和多角形，增殖活性和迁移能力均显著增加（P0.01），E-cadherin和p21蛋白表达显著下调（P0.05），而cyclin D1、cyclin E1、N-cadherin 和 vimentin 蛋白表达则显著上调（P0.05）。此外，与 WT 组相比，SETD4 KO 组细胞中 H3K4me2、H3K4me3、H3K9me1、H3K27me3、H3K79me2和H4K20me2水平显著降低（P0.05）。基因集富集结果显示，SETD4与细胞周期的功能和信号通路相关。结论：临床NPC组织中SETD4蛋白的表达减弱或缺失；SETD4表达减弱通过上调细胞周期相关蛋白促进NPC细胞增殖，通过诱导上皮-间充质转化而促进细胞迁移；SETD4影响NPC细胞中H3K4、H3K9、H3K27、H3K79和H4K20多个位点的甲基化。关键词 鼻咽癌；组蛋白甲基转移酶；含SET结构域蛋白4；细胞增殖；细胞迁移；基因集富集中图分类号 R363.2；R739.6 文献标志码 A doi：10.3969/j.issn.1000-4718.2023.02.008Effect of histone methyltransferase SETD4 on proliferation and migration of nasopharyngeal carcinoma cellsFENG Muyin1，ZHENG Axiu1，BAI Jianrong1，ZENG Yumei4，LUO Botao1，SHEN Zhihua1，JIE Wei1，2，3（1Department of Pathology，School of Basic Medical Sciences，Guangdong Medical University，Zhanjiang 524023，China；2Department of Oncology，the First Affiliated Hospital of Hainan Medical University，Haikou 570102，China；3Cancer Institute of Hainan Medical University，Haikou 571199，China；4Department of Pathology，Zhongshan Peoples Hospital，Zhongshan 528400，China.E-mail：；wei_）ABSTRACT AIM：To clarify the expression of histone methyltransferase SET-domain-containing protein 4（SETD4）in clinical nasopharyngeal carcinoma（NPC）tissues，and to analyze the effect of SETD4 on the proliferation and migration of NPC cells.METHODS：The protein levels of SETD4 in clinical 86 cases of NPC and 30 cases of control nasopharyngeal chronic inflammation（NPI）tissues was detected by immunohistochemistry.The SETD4 gene in CNE2 cells was knocked out based on CRISPR/Cas9 technology.The genotypes were identified by DNA sequencing and semi-quantita文章编号 1000-4718（2023）02-0259-10 收稿日期 2022-07-15 修回日期 2022-11-29*基金项目 广东省扬帆计划高层次人才项目（No.4YF16007G）通讯作者 申志华 Tel：0759-2388587；E-mail：；揭伟 Tel：0898-66968217；E-mail：wei_并列第一作者259tive RT-PCR.The morphology of cells was observed by inverted microscope，the proliferation activity of cells was analyzed by CCK-8 assay，and the changes in cell migration ability were assessed by Transwell assay.Proliferating cell nuclear antigen（PCNA），cell cycle-related proteins，markers of epithelial-mesenchymal transition and histone lysine methylationwere detected by Western blot.Finally，SETD4-associated functions and signaling pathways were enriched by bioinformatics.RESULTS：SETD4 protein was mainly localized in the nuclei of NPC cells，and the positive expression rate of SETD4 protein in NPC tissue was significantly lower than that in NPI control group（P0.01）.Based on CRISPR/Cas9 technology，the SETD4 gene in CNE2 was successfully knocked out，and homozygous cell lines were obtained.Compared with wild type（WT）cells，SETD4 knock out（KO）cells showed more obvious spindle and polygonal shapes，and the proliferation and migration ability were significantly increased（P0.01），the expression of E-cadherin and p21 protein were significantly down-regulated（P0.05），while cyclin D1，cyclin E1，N-cadherin，and vimentin protein expressions were significantly up-regulated（P0.05）.In addition，the levels of H3K4me2，H3K4me3，H3K9me1，H3K27me3，H3K79me2 and H4K20me2 were decreased in KO group compared with WT group（P0.05）.Gene sets enrichment results showed that SETD4 was associated with functions and signaling pathways related to cell cycle.CONCLUSION：The expression of SETD4 in clinical NPC tissues is generally attenuated or absent.The SETD4 mainly promotes NPC cell proliferation by mediating cell cycle-related proteins，and provokes cell migration by inducing epithelial-mesenchymal transition.The SETD4 mainly affects the methylation of H3K4，H3K9，H3K27，H3K79 and H4K20 in NPC cells.KEY WORDS nasopharyngeal carcinoma；histone methyltransferase；SET domain-containing protein 4；cell proliferation；cell migration；gene set enrichment鼻咽癌（nasopharyngeal carcinoma，NPC）是起源于鼻咽黏膜上皮的恶性肿瘤。遗传易感性和肿瘤微环境因素的胁迫与 NPC 发病和进展有很强的相关性1-2，而环境胁迫往往带来表观遗传修饰的改变3-4。组蛋白甲基化修饰被认为是表观遗传修饰中重要的方式之一5。含SETD结构域蛋白4（SET domain-containing protein 4，SETD4）作为组蛋白甲基化的重要成员之一，主要由位于羧基末端的SET结构域和位于氨基末端的底物结合结构域组成。其中SET结构域因其共同存在于果蝇的三个调节因子Su（var）3-9、E（z）、和Trithorax的羧基末端而得名，而含SET结构域的蛋白统称为 SET 家族蛋白6。SET家族蛋白普遍具有组蛋白和非组蛋白甲基化转移酶的功能，通过甲基化的修饰作用能够调控基因表达。课题组前期的研究提示 SETD4 对组蛋白 H3K4，H4K20的甲基化有影响7，但迄今对 SETD4基