犬乳腺肿瘤他莫昔芬耐药细胞中差异表达circRNAs的筛选与分析

Screening and Analysis of circRNAs Differentially Expressed in Tamoxifen-resistant Canine Mammary Gland Tumor Cells

【目的】筛选和分析环状RNA(circRNA)在犬乳腺肿瘤(CMGT)耐药中的作用,为肿瘤诊断和治疗提供新靶点。【方法】以CMGT细胞CHMm作为研究对象,首先通过浓度梯度法和浓度维持法构建CMGT他莫昔芬(TAM)耐药细胞系,然后采用CCK-8法检测CMGT细胞对TAM的敏感性,最后通过高通量测序技术筛选耐药细胞中差异表达的circRNAs,通过GO功能和KEGG信号通路分析其可能参与的生物过程。【结果】经过TAM持续刺激,CHMm形态变为长梭形,半数抑制浓度(IC 50)由4.07提高至13.75μmol/L,说明耐药细胞展现更强TAM抗性。测序结果显示,耐药细胞中差异表达的circRNAs 46个,GO功能分析涉及肿瘤细胞外基质、蛋白质类泛素化、细胞黏附和细胞迁移等过程,主要信号通路为Notch、Rap1、ErbB、FoxO和PI3K等通路。【结论】本研究成功构建犬乳腺肿瘤TAM耐药细胞系CHMm TAM,耐药细胞展现更强的增殖能力和TAM抗性。高通量筛选了亲本和耐药细胞中差异表达的circRNAs,耐药性产生可能通过Notch、Rap1和ErbB通路影响细胞信号转导、细胞泛素化、细胞转移等生物学过程,最终参与肿瘤耐药。

【Objective】The purpose of this study was to screen and analyze the role of circRNA in canine mammary gland tumor(CMGT)resistance,and provide a new target for tumor diagnosis and treatment.【Method】Taking CMGT cell CHMm as the research object,we firstly constructed CMGT tamoxifen(TAM)resistant cell lines by concentration gradient method and concentration maintenance method,then verified its resistance by CCK8,and finally screened differentially expressed circRNAs in drug-resistant cells by high-throughput sequencing technology.GO function and KEGG signaling pathway were used to analyze the biological processes in which it might be involved.【Result】The results showed that after continuous TAM stimulation,the CHMm morphology changed into long spindle shape,and the half inhibitory concentration(IC 50)increased from 4.07 to 13.75μmol/L,indicating that the drug-resistant cells showed stronger TAM resistance.Sequencing results showed that 46 circRNAs were differentially expressed in drug-resistant cells.GO function analysis involved the processes of tumor extracellular matrix,protein ubiquitination,cell adhesion and cell migration,and the major signaling pathways were Notch,Rap1,ErbB,FoxO and PI3K pathways.【Conclusion】In this study,the tamoxifen-resistant cell line CHMm TAM was successfully constructed in CMGT and the resistant cells showed stronger proliferation ability and TAM resistance.The differentially expressed circRNAs in parents and drug-resistant cells were screened in high throughput.It was also found that the generation of drug resistance might affect biological processes such as cell signal transduction,cell ubiquitination and cell metastasis through Notch,Rap1 and ErbB pathways,and ultimately participate in tumor drug resistance.