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| 隆纳霉素引起三阴性乳腺癌细胞周期阻滞基因表达谱及关键通路的生物信息学分析 |
| 薛佳兴△,孔杰△,卢小玲,孙筱,于豪冰,梁珍珍,徐尧,邓博文,焦炳华* |
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(海军军医大学(第二军医大学)基础医学院生物化学与分子生物学教研室, 上海 200433
△共同第一作者
*通信作者) |
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| 摘要: |
| 目的 利用生物信息学方法分析隆纳霉素对人三阴性乳腺癌细胞系细胞周期阻滞的作用机制。方法 利用隆纳霉素处理人三阴性乳腺癌细胞系MDA-MB-468细胞48、72 h，计算IC50，采用流式细胞术分析细胞周期变化。选取0.8 μmol/L隆纳霉素处理48 h的MDA-MB-468细胞和未加药处理的对照细胞进行转录组测序，测序数据经质量控制过滤后，使用DESeq2 1.16.1软件进行差异表达基因分析，并进行基因本体和京都基因与基因组百科全书功能分析及基因集富集分析，用STRING工具进行蛋白质相互作用网络分析及用AutoDock 1.5.6软件进行分子对接预测。结果 隆纳霉素干预MDA-MB-468细胞48、72 h的IC50分别为2.733和0.866 μmol/L，且流式细胞术分析显示隆纳霉素干预48 h有明显G2/M阻滞作用。隆纳霉素干预后共筛选出1 764个差异表达基因，主要定位于NF-κB介导的TNF-α通路和P53通路，G蛋白γ亚基7（GNG7）、G蛋白γ亚基11（GNG11）、C-X-C基序趋化因子配体8（CXCL8）、腺苷酸环化酶2（ADCY2）等基因可能是隆纳霉素作用基因网络中的关键节点。隆纳霉素与DNA分子、拓扑异构酶、基因表达调节蛋白[20S蛋白酶体β亚单位5（PSMB5）和含有SET结构域7的组蛋白赖氨酸甲基转移酶（SETD7）]有较好的结合及相互作用。结论 隆纳霉素具有与蝴蝶霉素类似的功能，其通过与DNA-拓扑异构酶Ⅰ复合物中的DNA分子结合发挥抗肿瘤活性；还可通过与基因表达调节蛋白PSMB5和SETD7结合影响GNG7、GNG11、CXCL8、ADCY2等基因的表达水平，从而上调NF-κB介导的TNF-α通路和P53通路，最终导致乳腺癌细胞发生G2/M阻滞，发挥抗肿瘤作用。 |
| 关键词: 隆纳霉素 蝴蝶霉素类似物 三阴性乳腺癌 细胞周期阻滞 |
| DOI：10.16781/j.0258-879x.2021.06.0585 |
| 投稿时间：2021-02-26修订日期：2021-04-30 |
| 基金项目:国家重点研发计划（2019YFC0312504）. |
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| Bioinformatics analysis of gene expression profile and key pathways of loongmycin-induced cell cycle arrest in triple-negative breast cancer |
| XUE Jia-xing△,KONG Jie△,LU Xiao-ling,SUN Xiao,YU Hao-bing,LIANG Zhen-zhen,XU Yao,DENG Bo-wen,JIAO Bing-hua* |
(Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Naval Medical University (Second Military Medical University), Shanghai 200433, China
△Co-first authors.
* Corresponding author) |
| Abstract: |
| Objective To analyze the mechanism of loongmycin-induced cell cycle arrest in triple-negative breast cancer cell line by bioinformatics. Methods The triple-negative breast cancer cell line, MDA-MB-468, was treated with loongmycin for 48 and 72 h. The half inhibition concentration (IC50) was calculated and the cell cycle was analyzed by flow cytometry. The MDA-MB-468 cells treated with 0.8 μmol/L loongmycin for 48 h and the control cells without drug treatment were selected for transcriptome sequencing. After the data were filtered by quality control, the differential gene expression was analyzed using DESeq2 1.16.1 software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional analyses and gene set enrichment analysis were performed. The protein-protein interaction network was analyzed by STRING and the docking prediction was performed using AutoDock 1.5.6 software. Results The IC50 values of MDA-MB-468 cells intervened by loongmycin for 48 and 72 h were 2.733 and 0.866 μmol/L, respectively. The results of flow cytometry showed that loongmycin arrested MDA-MB-468 cell line in G2/M phase after intervening for 48 h. A total of 1 764 differentially expressed genes were screened after intervention with loongmycin. The differentially expressed genes were mainly located in nuclear factor kappa B (NF-κB)-mediated tumor necrosis factor alpha (TNF-α) pathway and P53 pathway. G protein subunit gamma 7 (GNG7), G protein subunit gamma 11 (GNG11), C-X-C motif chemokine ligand 8 (CXCL8), adenylate cyclase 2 (ADCY2) and other genes may be the key nodes in the gene network of drug action. Loongmycin and DNA molecules, topoisomerase, gene expression regulator (proteasome 20S subunit beta 5 [PSMB5] and SET domain containing histone lysine methyltransferase 7 [SETD7]) had good combination and interaction functions. Conclusion Loongmycin has similar functions to rebeccamycin; it exerts anti-tumor activity by binding with DNA molecules in DNA-topoisomerase Ⅰ complex; and it can also affect the expression of GNG7, GNG11, CXCL8, ADCY2 and other genes by binding gene expression regulatory proteins PSMB5 and SETD7, and eventually up-regulate NF-κB-mediated TNF-α pathway and P53 pathway, leading to G2/M arrest in breast cancer cells, which plays an anti-tumor role. |
| Key words: loongmycin rebeccamycin analogs triple-negative breast neoplasms cell cycle arrest |
