目的 采用转录组学等技术探讨茶黄素-3,3′-双没食子酸酯(theaflavin-3,3′-digallate,TF3)对人骨肉瘤(HOS)细胞的抑制作用及其潜在机制。方法 分别使用不同浓度的TF3和体积分数0.05%的二甲基亚砜(dimethyl sulfoxide, DMSO)处理HOS细胞后,采用CCK-8(cell counting kit-8)法、结晶紫染色、集落形成实验和流式细胞术来检测TF3对HOS细胞增殖的影响; Hoechst33258染色实验及流式细胞术检测TF3对HOS细胞凋亡的影响;Western blot检测HOS细胞增殖和凋亡相关蛋白的水平;采用转录组测序检测TF3处理组和对照组HOS细胞的差异基因表达情况,并使用生物信息学分析潜在分子机制。根据转录组测序结果,采用实时荧光定量PCR法检测各组细胞中分泌粒蛋白Ⅱ(SCG2),蛋白酪氨酸激酶受体B1(EPHB1)和紧密连接蛋白7(CLDN7) mRNA 的表达。结果 与对照组相比,TF3处理组中HOS细胞的增殖能力明显受到抑制(P＜0.05),同时细胞凋亡率显著增高(P＜0.05),呈浓度依赖性;增殖相关蛋白增殖细胞核抗原(PCNA)和细胞增殖抗原(Ki67)及抗凋亡蛋白B细胞淋巴瘤/白血病-2(Bcl-2)表达量降低,凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶3(caspase-3)的水平下降,凋亡标志蛋白B细胞淋巴瘤/白血病-2相关X蛋白(Bax)、活化的半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3)和细胞色素C的表达量升高(P＜0.01);HOS细胞转录组测序结果显示,TF3处理组与对照组相比有809个差异表达基因(P-adjust＜0.05),其中596个上调,213个下调;基因本体论 (gene ontology,GO)主要富集于DNA复制的正调控、膜及蛋白结合等功能方面,京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)主要富集于缺氧诱导因子1信号通路、癌症通路及肿瘤坏死因子信号通路等包括凋亡在内与肿瘤相关的信号通路。与对照组相比,TF3处理组的SCG2, EPHB1, 和CLDN7 mRNA 表达水平下调(P＜0.05)。结论 TF3可能通过多靶点调控多种信号通路的活性,从而抑制HOS细胞增殖并促进其凋亡。
Abstract
OBJECTIVE To investigate the effects of theaflavin-3,3′-digallate (TF3) on proliferation and apoptosis of human osteosarcoma HOS cells and to explore its underlying mechanism through experiments including transcriptomics. METHODS Human osteosarcoma cells were treated with different concentrations of TF3 and 0.05% DMSO, respectively. Then, cell proliferation was detected by CCK-8 assay, crystal violet staining, colony formation assay and flow cytometry; cell apoptosis rate was detected by Hoechst33258 staining and flow cytometry. After treatment with of TF3 at different concentrations, the protein levels of the molecules related to proliferation and apoptosis of osteosarcoma HOS cells were determined by Western blot. The potential molecular mechanism of TF3 on HOS cells in vitro was revealed by transcriptomics and bioinformatic analysis. Real-time fluorescent quantitative PCR was performed in order to verify SCG2, EPHB1, and CLDN7 mRNA expression levels in cells based on results of transcriptome sequencing. RESULTS Compared with the control group, TF3 inhibited proliferation of osteosarcoma HOS cells and promoted apoptosis (P＜0.05) in a concentration-dependent manner. The expression levels of proliferation related proteins PCNA and Ki67 were all down-regulated by TF3 (P＜0. 01). TF3 decreased the expression of antiapoptotic protein Bcl-2 and apoptosis-related protein Caspase-3 (P＜0. 01). TF3 promoted the expression of apoptotic markers Bax, cleaved caspase-3 and cytochrome C (P＜0.01). Transcriptome analysis revealed 809 differential expressed genes(DEGs) after TF3 treatment in HOS cells, including 213 down-regulated and 596 up-regulated genes. The gene ontology (GO) analysis showed that the function of DEGs were mainly enriched in positive regulation of DNA replication, membrane, protein binding, etc. The Kyoto encyclopedia of genes and genome (KEGG) analysis showed that the function of DEGs were mainly enriched in apoptosis and tumor-related signaling pathways, including pathways in cancer, HIF-1 signaling pathway, TNF signaling pathway, etc. The expression of SCG2, EPHB1, and CLDN7 declined in comparison with the control group (P＜0.05). CONCLUSION TF3 inhibits the proliferation of osteosarcoma HOS cells, and also promote its apoptosis. Its molecular mechanism is potentially related to the regulation of multi-gene and multi-signaling pathway.
关键词
茶黄素-3,3′-双没食子酸酯 /
骨肉瘤 /
抗肿瘤 /
转录组测序 /
作用机制
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Key words
theaflavin-3,3′-digallate /
osteosarcoma /
antitumor /
transcriptomics sequencing /
mechanism
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参考文献
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