基于多数据平台探讨桂枝茯苓丸对乳腺癌的干预机制

Explore the Intervention Mechanism of Guizhi Fuling Pills on Breast Cancer Based on Multi-data Platform

关键词:  桂枝茯苓丸  乳腺癌  生物信息分析  PI3K/Akt信号通路  肿瘤微环境

Key words:  Guizhi Fuling pills  breast cancer  bioinformatics analysis  PI3K/Akt signaling pathway  tumor microenvironment

摘要:

目的 利用多数据平台与生物信息分析技术探讨桂枝茯苓丸对乳腺癌的干预机制，并通过细胞试验对分析结果进行验证。方法 从GEO数据库下载乳腺癌相关数据集，分析乳腺癌差异基因；从TCMSP数据库中筛选出桂枝茯苓丸的主要有效成分及作用靶基因；2组靶基因映射出桂枝茯苓丸治疗乳腺癌的关键靶基因。通过TCGA数据平台分析关键靶基因在乳腺癌中的表达，并通过TIMER、CPTAC Data Portal、Kaplan-Meier plotter、TISIDB、SangerBox等数据平台分析该表达与乳腺癌免疫微环境、免疫浸润水平、基因组异质性、免疫亚型和分子亚型以及不良预后的关系。通过Metascape分析平台对关键靶基因进行KEGG通路富集，寻找可能信号通路。最后采用血清药理学方法通过CCK-8试验、细胞划痕试验、Transwell小室试验及Western blotting技术在体外对分析结果进行验证。结果 桂枝茯苓丸中有效成分42个，对应靶点185个，与GEO数据库映射得到与乳腺癌治疗相关的关键靶点14个，经与TCGA中乳腺癌差异基因比对，得到桂枝茯苓丸治疗乳腺癌的6个关键靶基因。这6个靶基因表达与乳腺癌中6种免疫细胞水平、3个免疫微环境评分、乳腺癌免疫亚型及分子亚型都有很强的相关性（P<0.05），在基因组异质性相关性上也表现出了一致性，且与不良预后有密切的关系（P<0.05）。经KEGG富集分析发现，这6个基因主要富集在PI3K/Akt等信号通路上。细胞试验发现桂枝茯苓丸含药血清可以显著抑制乳腺癌HCC1937细胞的增殖、迁移与侵袭，还可降低HCC1937细胞中p-Akt/Akt、p-PI3K/PI3K蛋白相对表达，且作用呈剂量依赖性。结论 桂枝茯苓丸可能通过阻断PI3K/Akt信号通路，调节肿瘤免疫微环境实现对乳腺癌的干预作用。

Abstract:

OBJECTIVE To explore the intervention mechanism of Guizhi Fuling pills on breast cancer based on multi-data platform and bioinformation technology, and to verify the analysis results by cell test. METHODS The data set related to breast cancer was downloaded from GEO database to analyze the differential genes of breast cancer. The main active components and target genes of Guizhi Fuling pills were screened from TCMSP database. The key target genes of Guizhi Fuling pills in the treatment of breast cancer were mapped by the two groups of target genes. TCGA database was used to analyze the expression of key target genes in breast cancer. Through TIMER, CPTAC Data Portal, and Kaplan-Meier plotter, TISIDB, SangerBox and other data platforms, the relationships of the key genes expression and immune micro-environment, immune infiltration level, genomic heterogeneity, immune subtypes, molecular subtype, and poor prognosis were analyzed in breast cancer. KEGG pathway enrichment of key genes was performed searching for possible signaling pathways by Metascape analysis platform. Finally, CCK-8 assay, cell scratch assay, Transwell chamber assay and Western blotting technique were used to verify the results in vitro. RESULTS There were 42 active components and 185 targets in Guizhi Fuling pills, and 14 key targets related to the treatment of breast cancer were mapped with GEO database. Six key target genes for the treatment of breast cancer were obtained by comparison with breast cancer differential genes in TCGA. The expression of these 6 key genes was strongly correlated with the levels of 6 immune cells, 3 immune microenvironment scores, immune subtypes and molecular subtypes in breast cancer(P<0.05), also showed consistency in the correlation of genomic heterogeneity, and was closely associated with poor prognosis(P<0.05). KEGG enrichment analysis showed that these 6 genes were mainly enriched in PI3K/Akt signaling pathways. Cell test showed that Guizhi Fuling pills could significantly inhibit the proliferation, migration and invasion of HCC1937 cells, and also reduce the relative expression of p-Akt/Akt and p-PI3K/PI3K proteins in HCC1937 cells in a dose-dependent manner. CONCLUSION Guizhi Fuling pills may interfere with breast cancer by blocking PI3K/Akt signaling pathway and regulating the tumor immune microenvironment.