摘要
目的:探讨知母皂苷AⅢ(TAⅢ)对非小细胞肺癌(NSCLC)A549细胞增殖、迁移和侵袭的影响机制。方法:用不同浓度的TAⅢ处理A549细胞后,CCK-8法检测TAⅢ对A549细胞的细胞毒性。体外培养A549细胞,分为对照组(Control组)、TAⅢ组(20μmol·L^(-1))、TAⅢ+anti-NC组、TAⅢ+anti-miR-129-5p组,实时荧光定量PCR(RT-qPCR)检测细胞中miR-129-5p、信号转导和转录激活因子3(STAT3)mRNA表达,蛋白质印迹(Western blot)检测STAT3及其磷酸化蛋白(p-STAT3)表达,平板克隆实验检测细胞增殖活力,Transwell实验检测细胞迁移和侵袭能力。双荧光素酶实验验证miR-129-5p和STAT3的靶向关系。BALB/c裸鼠移植瘤实验检测TAIII在体内的抗肿瘤活性,免疫组织化学(IHC)法检测肿瘤组织Ki-67、p-STAT3表达。结果:低浓度TAⅢ(1,5μmol·L^(-1))对A549细胞存活率无显著影响(P>0.05),高浓度TAⅢ(10,20,30,60μmol·L^(-1))呈浓度依赖性降低A549细胞存活率(P<0.05)。TAⅢ可显著升高A549细胞中miR-129-5p水平,降低STAT3 mRNA和蛋白以及p-STAT3蛋白水平,抑制A549细胞的克隆形成和迁移、侵袭能力(P<0.05);双荧光素酶实验证实STAT3是miR-129-5p的潜在靶标(P<0.05);体内实验证实,TAⅢ可抑制裸鼠体内移植瘤生长(P<0.05);下调miR-129-5p可增强STAT3表达和活化,减弱TAⅢ对NSCLC细胞恶性表型和体内移植瘤生长的抑制作用(P<0.05)。结论:TAⅢ可能通过上调miR-129-5p,抑制STAT3表达和活化,进而抑制A549细胞增殖、迁移和侵袭。
Objective: To investigate the action mechanism of timosaponin AⅢ(TAⅢ) on the proliferation, migration and invasion of non-small cell lung cancer(NSCLC) A549 cells. Methods: After A549 cells were treated with different concentrations of TAⅢ, the cytotoxicity of TAⅢ on A549 cells was detected by CCK-8 method. A549 cells were cultured in vitro and divided into control group, TAⅢ group(20 μmol·L^(-1)), TAⅢ+anti-NC group and TAⅢ+anti-miR-129-5 p group. Real-time quantitative PCR(RT-qPCR) was used to detect the expression of miR-129-5 p, signal transducer and activator of transcription 3(STAT3) mRNA in cells, Western blot was used to detect the expression of STAT3 and its phosphorylated protein(p-STAT3), plate cloning assay was used to detect cell proliferation activity, and Transwell assay was used to detect cell migration and invasion abilities. The dual-luciferase experiment was used to verify the targeting relationship between miR-129-5 p and STAT3. The antitumor activity of TAⅢ in vivo was detected by BALB/c nude mice xenograft experiments, and the expression of Ki-67 and p-STAT3 in tumor tissue was detected by immunohistochemistry(IHC). Results: Low concentrations of TAⅢ(1, 5 μmol·L^(-1)) had no significant effect on the viability of A549 cells, while high concentrations of TAⅢ(10, 20, 30, 60 μmol·L^(-1)) decreased the viability of A549 cells in a concentration-dependent manner(P<0.05). TAⅢ could significantly increase the level of miR-129-5 p in A549 cells, reduce the levels of STAT3 mRNA and protein and p-STAT3 protein, and inhibit the clone formation, migration and invasion abilities of A549 cells(P<0.05);dual luciferase experiments confirmed that STAT3 was a potential target of miR-129-5 p(P<0.05);in vivo experiments confirmed that TAⅢ could inhibit the growth of transplanted tumor in nude mice(P<0.05);down-regulation of miR-129-5 p enhanced the expression and activation of STAT3, and attenuated the inhibitory effects of TAⅢ on the malignant phenotype of NSCLC cells and the growth of xenografts in vivo(P<0.05). Conclusion: TAⅢ may inhibit the proliferation, migration and invasion of A549 cells by up-regulating miR-129-5 p and inhibiting the expression and activation of STAT3.
作者
段锦华
雷蕾
李婕
高霞
Duan Jinhua;Lei Lei;Li Jie;Gao Xia(Department of Pathology,The People’s Hospital of Pengzhou,Sichuan Pengzhou 611930,China;Department of Oncology,The Sixth People’s Hospital of Chengdu)
出处
《中国药师》
CAS
2022年第12期2171-2176,共6页
China Pharmacist
基金
成都市卫生健康委员会资助项目(编号:2021110)。
