南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (8): 1297-1305.doi: 10.12122/j.issn.1673-4254.2023.08.05

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脱醇红酒抑制肝细胞癌的发生和发展：基于诱导细胞周期的阻滞和凋亡

兰 玉，王凯风，蓝智贤，周何琪，孙 剑   

  1. 器官衰竭防治国家重点实验室，广东省病毒性肝炎研究重点实验室，南方医科大学南方医院感染内科，广东 广州 510515
  • 出版日期:2023-08-20 发布日期:2023-09-13

Dealcoholized red wine inhibits occurrence and progression of hepatocellular carcinoma possibly by inducing cell cycle arrest and apoptosis

LAN Yu, WANG Kaifeng, LAN Zhixian, ZHOU Heqi, SUN Jian   

  1. State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
  • Online:2023-08-20 Published:2023-09-13

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摘要： 目的 探讨脱醇红酒对肝细胞癌（HCC）发生发展的抑制作用，并初步探索其可能的机制。方法 不同浓度的脱醇红酒（0、5、10、25、50和100 μL/mL）处理人肝癌细胞（Huh7，HepG2和SK-Hep-1）后，采用CCK-8和克隆形成实验分别检测各组细胞的增值活性和克隆形成能力。构建裸鼠皮下瘤模型，分为实验组（脱醇红酒，300 μL/d）和对照组，干预4周后对比两组肿瘤体积。构建化学诱导小鼠肝癌模型，分为实验组（脱醇红酒，300 μL/d）和对照组，干预6周后对比两组肝脏肿瘤数量、最大肿瘤直径和肝体比。脱醇红酒（60 μL/mL）处理48 h后，利用RNA-seq对Huh7细胞转录组测序，基因集富集分析（GSEA）观察基因集及通路的变化；流式细胞术检测脱醇红酒（60 μL/mL）干预后Huh7细胞周期和凋亡变化。结果 在体外，脱醇红酒呈浓度和时间依赖性地抑制肝癌细胞的增殖活性，呈浓度依赖性地抑制其克隆形成能力。在体内，相比于对照组，脱醇红酒显著抑制实验组裸鼠皮下瘤的体积（P<0.05），也能显著抑制实验组化学诱导肝癌小鼠的肝脏肿瘤数量（P<0.001）、最大肿瘤直径(P<0.05）和肝体比（P<0.01）。RNA-seq结果显示，脱醇红酒干预后Huh7细胞内有634个基因上调和478个基因下调（|log2FC|≥2，Q≤0.05）。基因集富集分析发现，脱醇红酒能够诱导细胞周期通路相关基因集显著下调（包括E2F Targets、G2M Checkpoint、MYC Targets等）和细胞凋亡通路相关基因集上调；流式细胞检测结果表明，脱醇红酒的干预使Huh7细胞G1期阻滞，同时诱导细胞凋亡。结论 脱醇红酒对HCC的发生发展具有抑制作用，其机制可能与细胞周期和凋亡相关通路介导的肝癌细胞G1期阻滞和细胞凋亡有关。

关键词: 脱醇红酒, 肝细胞癌, 多酚, RNA-seq, 细胞周期, 细胞凋亡

Abstract: Objective To investigate the inhibitory effect of dealcoholized red wine (DRW) on occurrence and progression of hepatocellular carcinoma (HCC) and explore its possible mechanisms. Methods Three HCC cell lines (Huh7, HepG2 and SK-Hep-1) treated with 5, 10, 25, 50 and 100 μL/mL DRW were examined for changes in proliferation and colony formation ability using CCK-8 assay and colony formation assay. A nude mouse model bearing subcutaneous HCC xenograft was used to test the effect of 300 μL/day DRW for 4 weeks on tumor growth. The inhibitory effect of 300 μL/day DRW for 6 weeks on tumor growth was also observed in a mouse model of chemically induced HCC by examining the tumor number, largest tumor diameter and the liver/body ratio. RNA-seq technique was used for transcriptome sequencing of Huh7 cells treated with DRW (60 μL/mL) for 48 h, and gene-set enrichment analysis (GSEA) was performed to identify the changes in genes and pathways. Flow cytometry assay was used to analyze the changes in cell cycle and apoptosis of the cells. Results DRW inhibited the proliferation of the HCC cell lines in a concentration- and time-dependent manner, and concentration-dependently inhibited colony formation of the cells. Treatment with DRW significantly reduced the volume of subcutaneous tumor xenograft in the tumor-bearing nude mice (P<0.05), and lowered the number of tumors (P<0.001), the largest tumor diameter (P<0.05) and the liver/body ratio (P<0.01) in mice with chemically induced HCC. RNA-seq showed that 634 genes were significantly up-regulated and 478 were down-regulated in Huh7 cells after treatment with DRW. Gene-set enrichment analysis revealed that DRW significantly down- regulated cell cycle- related pathways (E2F Targets, G2M Checkpoint and MYC Targets) and up-regulated apoptosis pathways. Flow cytometry assay showed that DRW induced cell cycle arrest in G1 phase and apoptosis of Huh7 cells. Conclusion DRW inhibits the occurrence and progression of HCC, and this effect is mediated possibly by inducing cell cycle arrest and apoptosis.

Key words: dealcoholized red wine, hepatocellular carcinoma, polyphenol, RNA-seq, cell cycle, apoptosis