Objective: To analyze the differential expression of silent information regulator transcript-1 (SIRT1) in tissues and cells of nasopharyngeal carcinoma (NPC), to explore the effects of SIRT1 on the proliferation and migration of NPC cells, as well as the effects on and mechanisms of lipid metabolism in NPC cells. Methods: Experimental subjects: In this study, tissue specimens were obtained from patients who visited the Department of Otolaryngology and performed nasopharyngeal tissue biopsy in the Affiliated Hospital of Nantong University from 2019 to 2020. Among them, 6 cases were male, 6 cases were female, age range: 27-72 years old, including 7 cases of NPC diagnosed by pathology and 5 cases of normal nasopharyngeal mucosa. Experimental methods and outcome measures: Western Blot and quantitative real time polymerase chain reaction (qRT-PCR) were used to detect the protein and mRNA levels of SIRT1. CNE2 cell line was selected for subsequent experiments. Cell viability and migratory ability were evaluated by CCK8, wound healing and Transwell assays respectively. Animal xenograft tumor model was used to explore the role of SIRT1 inhibitor Ex527 on tumor growth in nude mice. Oil red and Bodipy were used to stain intracellular lipids. For the mechanical investigation, the interactions between SIRT1 and hypoxia inducible factor-1α (HIF-1α) were analyzed by immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP). Finally, statistical analysis was performed by SPSS 26.0 software, P<0.05 was considered statistically significant. Results: The levels of SIRT1 protein (1.005±0.168) and mRNA (5.829±2.395) in NPC tissues were higher than those in normal nasopharyngeal mucosa (0.181±0.042,1.995±1.605). Differences were statistically significant (t values were 6.438 and 2.759, both P<0.05). The mRNA and protein levels of CNE1, CNE2, 5-8F and 6-10B cell lines were also higher than those in normal nasopharynx epithelial cell line NP69. Besides, overexpression of SIRT1 correlated with the proliferation and migration of NPC cells. The tumorigenesis ability of nude mice in the Ex527 group was lower than that in the control group. The low SIRT1 expression reduced the protein level of the key enzymes of liposynthesis in NPC cells, improved the expression of lipolysis enzymes, while HIF-1α overexpression promoted lipid synthesis enzymes in NPC cells. SIRT1 inhibited HIF-1α transcription by enhancing deacetylation levels. The binding ability of HIF-1α to SIRT1 promoter regions decreased when NPC cells were hypoxic. Conclusions: SIRT1 promotes the proliferation, migration and lipid metabolism of nasopharyngeal carcinoma cells, which might be expected to provide new theoretical basis for prognosis judgment and gene therapy.
目的： 分析沉默信息调节因子1（silent information regulator transcript-1，SIRT1）在鼻咽癌（nasopharyngeal carcinoma，NPC）组织和细胞中的表达差异，探讨SIRT1对NPC细胞增殖和迁移的影响，研究SIRT1对NPC细胞脂质代谢的影响及调控机制。 方法： 实验对象：组织标本来源于2019—2020年间就诊于南通大学附属医院耳鼻咽喉科并进行鼻咽部组织活检的患者，其中男6例，女6例，年龄27~72岁；其中经病理确诊的鼻咽癌7例，正常鼻咽部黏膜5例。另NPC细胞系CNE1、CNE2、5-8F和6-10B以及永生化的正常鼻咽上皮细胞NP69为中南大学湘雅医院和中山大学肿瘤防治中心赠送。实验方法和观察指标：采用免疫蛋白印迹法（Western Blot）和实时荧光定量反转录聚合酶链反应（quantitative real time polymerase chain reaction，qRT-PCR）分别检测鼻咽癌组织和正常鼻咽部黏膜组织中SIRT1蛋白和mRNA水平。后续实验选取CNE2为实验的主要细胞，通过CCK8试剂盒检测细胞活力，利用划痕实验和Transwell系统检测细胞迁移能力。采用裸鼠异体肿瘤模型研究SIRT1抑制剂Ex527对体内肿瘤生长的影响。通过油红和氟化硼二吡咯（Bodipy）染料标记细胞内脂滴。在机制研究方面，利用免疫沉淀（immunoprecipitation，IP）和染色质免疫共沉淀（chromatin immunoprecipitation，ChIP）分析SIRT1与缺氧诱导因子1α（hypoxia inducible factor-1α，HIF-1α）的相互作用。所有数据采用SPSS 26.0软件进行统计分析，以P<0.05为差异具有统计学意义。 结果： NPC组织中SIRT1蛋白（1.005±0.168）和mRNA（5.829±2.395）水平均高于正常鼻咽黏膜组织（0.181±0.042、1.995±1.605），差异有统计学意义（t值分别为6.438、2.759，P值均<0.05）。NPC细胞系CNE1、CNE2、5-8F和6-10B中SIRT1的mRNA和蛋白水平也高于永生化的正常鼻咽上皮细胞NP69。SIRT1的过表达促进了NPC细胞的增殖和迁移能力。注射Ex527组的裸鼠成瘤能力低于对照组。SIRT1的低表达降低了NPC细胞脂质合成关键酶的蛋白表达水平，提高了脂质分解酶的蛋白表达水平，HIF-1α过表达促进了NPC细胞脂质合成酶的蛋白表达。SIRT1通过增强HIF-1α的去乙酰化水平抑制HIF-1α转录。HIF-1α与SIRT1启动子区域的结合能力在NPC细胞缺氧时下降。 结论： SIRT1促进NPC的增殖、迁移和脂质代谢，有望为预后判断和基因治疗提供新的参考。.
Keywords: Hypoxia inducible factor-1α; Lipid metabolism; Nasopharyngeal carcinoma; Silent information regulator transcript-1.