南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (12): 1763-1772.doi: 10.12122/j.issn.1673-4254.2021.12.02

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天然虾青素治疗肾透明细胞癌的作用机制：基于网络药理学与生物信息学方法

高俊杰，杨丹丹，曹若雪，潘雪珊，夏 俊   

  1. 蚌埠医学院检验医学院肿瘤基础研究与临床检验诊断重点实验室，生化与分子生物学教研室，安徽 蚌埠 233030；连云港市第二人民医院检验科，江苏 连云港 222000
  • 出版日期:2021-12-20 发布日期:2022-01-04

Therapeutic mechanism of natural astaxanthin against renal clear cell carcinoma based on network pharmacology and bioinformatics

GAO Junjie, YANG Dandan, CAO Ruoxue, PAN Xueshan, XIA Jun   

  1. Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis, Department of Biochemistry and Molecular Biology, School of Laboratory Medicine, Bengbu Medical College, Bengbu 233030, China; Department of Laboratory Medicine, Second People's Hospital of Lianyungang, Lianyungang 222000, China
  • Online:2021-12-20 Published:2022-01-04

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摘要： 目的 基于网络药理学和生物信息学研究天然虾青素（AST）治疗肾透明细胞癌（KIRC）的分子作用机制。方法 利用PharmMapper数据库检索AST的作用靶点，TCGA数据库获得并筛选KIRC癌组织和癌旁组织中的差异基因，Cytoscape软件对靶基因进行分析并构建“药物-靶点”网络图，String数据库构建可视化PPI网络，对核心作用靶点进行GO富集分析。接着，对筛选出的核心靶点胎盘生长因子（PGF）进行单基因生物信息学分析，采用CCK-8法验证AST对KIRC细胞活性的影响，分子对接技术验证AST与PGF结合情况，RT-qPCR和Western blot验证AST对靶基因mRNA和蛋白表达的影响。结果 分析得到AST作用靶点278个，KIRC相关作用靶点1081个，AST治疗KIRC的核心作用靶点7个，其中核心靶点PGF在KIRC中表达增高（P<0.001）且与预后不良相关（HR=1.37，P=0.043）；分子对接显示AST与PGF的结合能为-5.43 kcal/mol；CCK-8显示当AST浓度为50 μmol/L时即可以抑制KIRC细胞的增殖且AST浓度越高抑制作用越显著；RT-qPCR结果显示，AST处理KIRC后PGF的mRNA表达水平显著降低；Western blot结果显示，经AST处理后KIRC中PGF的蛋白表达受到了明显抑制。结论 AST可以抑制KIRC的增殖并抑制其细胞中PGF的表达。

关键词: 网络药理学；生物信息学；天然虾青素；肾透明细胞癌；胎盘生长因子

Abstract: Objective To explore the molecular mechanism by which natural astaxanthin (AST) inhibits renal clear cell carcinoma (KIRC) based on network pharmacology and bioinformatics. Methods PharmMapper database was used to retrieve the targets of natural astaxanthin, and TCGA database was used to identify the differentially expressed genes (DEGs) in KIRC and adjacent tissues. The target genes of AST was analyzed using Cytoscape software to construct the "drug-target" network diagram. The visual protein-protein interaction (PPI) network was constructed using String database, and GO enrichment analysis of the core targets was performed. Single gene bioinformatics was performed to verify the screened core target of AST, namely placental growth factor (PGF). The effect of natural AST on the viability of KIRC cells was tested using CCK-8 method, and the binding between natural AST and PGF was assessed with molecular docking technology. The effect of natural AST on the mRNA and protein expression of the target genes was analyzed using RT-qPCR and Western blotting. Results We identified 278 candidate targets of AST, 1081 KIRC-related targets, and 7 core targets involved in the therapeutic mechanism of AST against KIRC. Among these 7 core targets, PGF showed significantly upregulated expression in KIRC (P<0.001) in correlation with a poor prognosis (HR=1.37, P=0.043). Molecular docking showed that the binding energy of AST and PGF was -5.43 kcal/mol. CCK-8 assay showed that AST at the concentration of 50 μmol/L was capable of inhibiting the proliferation of KIRC cells, and a higher concentration resulted in a stronger inhibitory effect. The results of RT-qPCR and Western blotting showed that AST treatment significantly reduced the expression of PGF at both the mRNA and protein levels in KIRC cells. Conclusion Natural AST can suppress the proliferation of KIRC and inhibit the expression of PGF in the cells.

Key words: network pharmacology; bioinformatics; natural astaxanthin; renal clear cell carcinoma; placental growth factor