目的: 研究知母皂苷A-Ⅲ (timosaponin A-Ⅲ,TAⅢ)对非小细胞肺癌(non-small cell lung cancer,NSCLC)生长的影响及其作用机制。方法: 采用不同浓度TAⅢ(5、10、15、20、25、30 μmol/L)处理非小细胞肺癌细胞A549,使用CCK8法检测细胞活力。以定量逆转录聚合酶链式反应(qRT-PCR)检测二磷酸腺苷核糖化因子鸟苷酸激酶1-内含子转录因子1 (adenosine diphosphate ribosylation factor guanylate kinase 1-intronic transcript 1,ASAP1-IT1)、Yes相关蛋白1(Yes-associated protein 1,YAP1)在组织及细胞中的表达。以EDU、流式细胞术和Western blot检测ASAP1-IT1、DNA(胞嘧啶-5-)-甲基转移酶3β(DNA (cytosine-5-)-methyltransferase 3 beta,DNMT3b)、YAP1对细胞增殖情况、细胞凋亡率及其相关基因(Bax和caspase-3)与抗凋亡基因Bcl2的影响。亚硫酸氢盐测序聚合酶链式反应(Bisulfite sequencing PCR, BSP)分析检测ASAP1-IT1与YAP1的关系。RNA免疫沉淀法(RNA binding protein immunoprecipitation,RIP)和qRT-PCR法检测ASAP1-IT1与DNMT3b之间的相互作用。免疫共沉淀实验(Co-immunoprecipitation,Co-IP)检测DNMT3b与YAP1之间的关系。采用qRT-PCR方法检测TAⅢ对ASAP1-IT1/ DNMT3b /YAP1轴相关基因表达的影响。结果: ASAP1-IT1和YAP1在NSCLC组织和A549细胞中表达上调,而DNMT3b表达下调,加入TAⅢ可逆转这些效应。沉默ASAP1-IT1和YAP1、过表达DNMT3b或应用TAⅢ可增加A549细胞凋亡率和细胞凋亡相关指数。TAⅢ有逆转沉默或过表达ASAP1-IT1、DNMT3b和YAP1的作用。最后发现ASAP1-IT1与DNMT3b相互作用,而DNMT3b与YAP1相互作用。结论: ASAP1-IT1/DNMT3b/YAP1轴通过调节细胞增殖和凋亡促进NSCLC进展。TAⅢ通过调节ASAP1-IT1/DNMT3b/YAP1轴抑制肺癌细胞增殖,促进其凋亡。
Objective: To explore the effect and mechanism of Timosaponin A-Ⅲ (TAⅢ) on NSCLC growth. Methods: A549 cells was processed with different concentrations of TAIII (5, 10, 15, 20, 25, 30 μmol/L), the cell viability was measured by CCK-8. qRT-PCR was used to detect the expressions of Adenosine diphosphate ribosylation factor guanylate kinase 1-intronic transcript 1(ASAP1-IT1) and Yes-associated Protein 1(YAP1) in tissues and cells. EDU, flow cytometry and Western blot were used to detect ASAP1-IT1, DNA (cytosine-5-)-methyltransferase 3β (DNMT3b), YAP1 and their effects on cell proliferation, apoptosis rate and the influence of apoptosis related genes (Bax and caspase-3) and anti-apoptotic gene Bcl2. Bisulfite sequencing PCR (BSP) was used to analysis the relationship between ASAP1-IT1 and YAP1. RNA immunoprecitation (RIP) and qRT-PCR assays were used to analyze the interaction between ASAP1-IT1 and DNMT3b. Co-immunoprecipitation (Co-IP) was used to confirm the interaction between YAP1 and DNMT3b. qRT-PCR method was used to analyze the effect of TAⅢ on the expression of ASAP1-IT1/DNMT3b/YAP1 axis-related genes. Results: ASAP1-IT1 and YAP1 was up-regulated in NSCLC tissues and cancer cells, and DNMT3b was down-regulated in NSCLC tissues and cancer cells, the levels of ASAP1-IT1, YAP1 and DNMT3b were reversed after using TAⅢ. Silencing of ASAP1-IT1 and YAP1, overexpression of DNMT3b or taking TAⅢ significantly increased apoptosis rate of A549 cells and apoptosis related indexes. TAⅢ reversed the silencing or overexpression of ASAP1-IT1, DNMT3b and YAP1. Furthermore, ASAP1-IT1 was found to interact with DNMT3b, and DNMT3b and YAP1 were interacted. Conclusion: ASAP1-IT1/DNMT3b/YAP1 axis promotes NSCLC progression by regulating cell proliferation and apoptosis. TAⅢ suppresses proliferation and improve apoptosis of lung cancer cells by regulating ASAP1-IT1/DNMT3b/ YAP1 axis.
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