Department of Clinical Laboratory, The PLA General Hospital, Beijing, 100853, China.
Department of Clinical Laboratory of Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, 100091, China.
Sci China Life Sci. 2018 May;61(5):550-558. doi: 10.1007/s11427-016-9050-6. Epub 2017 Nov 7.
We previously demonstrated that matrine could inhibit the proliferating, migrating, as well as invading processes of both PC-3 and DU145 cells. However, the underlying molecular mechanisms have not yet been clearly defined. In this study, using various techniques such as high throughput sequencing technology, bioinformatics, quantitative real-time PCR, and immunoblot analysis, we aimed to understand whether matrine serves as a novel regulator of FOXO and PI3K-AKT signaling pathway. DU145 and PC-3 cell lines were cultured for 24 h in vitro. Cells were treated with either matrine or control serum for 48 h, followed by extraction of total RNA. The RNA was sequenced using HiSeq 2500 high-throughput sequencing platform (Illumina). A gene library was established and quality analysis of read data carried out. Integrated database from the website DAVID was used to analyze Gene Ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathway of differential genes was used for pathway analysis, screening for fold differences of more than two times. The FOXO and PI3K-AKT signaling pathways were screened, and expression levels of mRNA and core protein detected by real-time PCR and immunoblotting, respectively. High throughput sequencing and GO analysis revealed that differentially expressed genes before and after treatment played an important role in cell metabolic process, growth process, anatomical structure formation, cellular component organization, and biological regulation. KEGG signal pathway analysis revealed that FOXO and PI3K-AKT signal pathways had a significant difference between before and after matrine-treated androgen-independent prostate cancer cells PC-3 and DU145. Real-time PCR showed that matrine treatment led to a significant increase in the expression levels of FOXO1A, FOXO3A, FOXO4, and FOXO6 in DU145 and PC-3 cells (P<0.01 or P<0.05), whereas the PI3K expression levels decreased (P<0.01). Similarly, immunoblotting revealed a significant increase (P<0.05) in the expression levels of FOXO1A FOXO3A, FOXO4, and FOXO6 in both PC-3 and DU145 cells, whereas PI3K expression levels decreased (P<0.05). Matrine had a broad regulating effect on the mRNA expression profiles of both PC-3 and DU145 cells. Matrine may inhibit cell proliferation, migration, as well as invasion, and induce apoptosis in both PC-3 and DU145 cells through FOXO and PI3K-AKT signaling pathways. Matrine could therefore be used as a complementary drug to present chemotherapeutic agents, for treating androgen-independent prostate cancer.
我们之前的研究表明，苦参碱可以抑制 PC-3 和 DU145 细胞的增殖、迁移和侵袭过程。然而，其潜在的分子机制尚未明确。在这项研究中，我们使用高通量测序技术、生物信息学、实时定量 PCR 和免疫印迹分析等多种技术，旨在了解苦参碱是否可以作为 FOXO 和 PI3K-AKT 信号通路的新型调节剂。将 DU145 和 PC-3 细胞系在体外培养 24 小时。用苦参碱或对照血清处理细胞 48 小时，然后提取总 RNA。使用 HiSeq 2500 高通量测序平台（Illumina）对 RNA 进行测序。建立基因文库，并对读数据进行质量分析。使用来自 DAVID 网站的集成数据库分析差异基因的基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路，用于通路分析，筛选倍数变化超过两倍的基因。筛选 FOXO 和 PI3K-AKT 信号通路，并通过实时 PCR 和免疫印迹分别检测 mRNA 和核心蛋白的表达水平。高通量测序和 GO 分析表明，处理前后差异表达的基因在细胞代谢过程、生长过程、解剖结构形成、细胞成分组织和生物调节中起着重要作用。KEGG 信号通路分析表明，苦参碱处理后的雄激素非依赖性前列腺癌 PC-3 和 DU145 细胞的 FOXO 和 PI3K-AKT 信号通路有显著差异。实时 PCR 显示苦参碱处理后，DU145 和 PC-3 细胞中 FOXO1A、FOXO3A、FOXO4 和 FOXO6 的表达水平显著升高（P<0.01 或 P<0.05），而 PI3K 的表达水平降低（P<0.01）。同样，免疫印迹也显示，PC-3 和 DU145 细胞中 FOXO1A、FOXO3A、FOXO4 和 FOXO6 的表达水平显著升高（P<0.05），而 PI3K 的表达水平降低（P<0.05）。苦参碱对 PC-3 和 DU145 细胞的 mRNA 表达谱有广泛的调节作用。苦参碱可能通过 FOXO 和 PI3K-AKT 信号通路抑制 PC-3 和 DU145 细胞的增殖、迁移和侵袭，并诱导细胞凋亡。因此，苦参碱可以作为一种补充药物与现有的化疗药物联合使用，用于治疗雄激素非依赖性前列腺癌。
