基于生物信息学及体外实验研究大黄素治疗胰腺癌的靶点

The target of emodin in the treatment of pancreatic cancer based on bioinformatics combined with in vitro experiments

  • 摘要: 目的:运用生物信息学及细胞实验方法研究大黄素治疗胰腺癌的靶点。方法:从基因表达综合数据库(GEO)获取胰腺癌转录组数据集GSE62452、GSE28753,通过中药系统药理学数据库和分析平台(TCMSP)以及PharmMapper Server协同分析获得大黄素作用靶点,将两者靶基因进行交叉筛选,获得大黄素在胰腺癌中的潜在靶点基因。通过DAVID数据库对靶点基因做KEGG、GO功能富集分析,通过GEPIA数据库对潜在靶点基因做差异表达和生存分析。体外培养胰腺癌细胞,CCK-8法检测不同浓度大黄素对胰腺癌细胞增殖的影响。将细胞分为正常对照组和不同浓度大黄素组,实时荧光定量PCR(RT-qPCR)检测纤溶酶原激活剂(PLAU)、酪氨酸激酶受体(MET)、环氧化物水解酶2(EPHX2)、基质金属肽酶1(MMP1)mRNA表达,western blotting检测EPHX2蛋白表达。结果:获得大黄素和胰腺癌17个共同靶点基因。功能富集分析显示,靶基因主要富集在细胞外空隙、细胞外来体、丝氨酸内切酶活性、蛋白质水解、钙离子结合等通路。差异表达分析显示:胰腺癌组织与正常组织MMP1、EPHX2、PLAU、MET基因表达比较,差异有统计学意义(P<0.05),生存分析显示,MMP1、EPHX2、PLAU、MET、TTR基因在胰腺癌中低表达组与高表达组之间差异具有统计学意义(P<0.05)。体外实验结果显示,随着大黄素浓度的增加,胰腺癌细胞存活率降低(P<0.05)。与正常对照组相比,20μmol/L和40μmol/L大黄素组PLAU、MMP1、MET、EPHX2 mRNA表达水平升高,EPHX2蛋白表达水平升高(均P<0.05)。结论:EPHX2可能是大黄素治疗胰腺癌的潜在靶点。

     

    Abstract: Objective: To study the target of emodin in the treatment of pancreatic cancer by bioinformatics and cell experiments. Methods: The pancreatic cancer transcriptome datasets GSE62452 and GSE28753 were obtained from the Gene Expression Omnibus(GEO) database, and the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and PharmMapper Server were used to obtain the target of emodin. The target genes of the two were cross-screened to obtain the potential target genes of emodin in pancreatic cancer. The DAVID database was used for KEGG and GO functional enrichment analysis of target genes, and GEPIA database was used for differential expression and survival analysis of potential target genes. Pancreatic cancer cells were cultured in vitro and cell counting kit-8(CCK-8) assay was used to detect the effect of different concentrations of emodin on the proliferation of pancreatic cancer cells. The cells were divided into normal control group and different concentrations of emodin group. The real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the mRNA expression of plasminogen activator urokinase(PLAU), tyrosine kinase receptor(MET), epoxide hydrolase 2(EPHX2), and matrix metalloproteinase 1(MMP1). Western blotting was used to detect the expression of EPHX2 protein. Results: Seventeen common target genes of emodin and pancreatic cancer were obtained. Functional enrichment analysis showed that the target genes were mainly enriched in extracellular space, extracellular vesicle, serine endopeptidase activity, protein hydrolysis, calcium ion binding and other pathways. Differential expression analysis showed that there were significant differences in MMP1, EPHX2, PLAU, MET, and TTR gene expression between pancreatic cancer tissues and normal tissues(P<0.05). Survival analysis results showed that MMP1, EPHX2, PLAU, MET, TTR genes had statistically significant differences between the low expression group and the high expression group in pancreatic cancer(P<0.05). The results of in vitro experiments showed that the survival rate of pancreatic cancer cells was decreased with the increase of emodin concentration(P<0.05). Compared with the normal control group, the mRNA expression levels of PLAU, MMP1, MET, EPHX2 genes in the 20 umol/LEmo group and 40 umol/LEmo group were increased, and the expression level of EPHX2 protein was increased(all P<0.05). Conclusion: EPHX2 may be the target of emodin in pancreatic cancer.

     

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