黄芪-白花蛇舌草调控Sp1-miR-582-3p-p27轴抑制肺腺癌增殖分子机制  

Molecular Mechanism of Astragali Radix and Hedyotis diffusa Regulating Sp1-miR-582-3p-p27 Axis to Inhibit Lung Adenocarcinoma Proliferation

作　　者：孙海鹏 许露凡 刘学 刘思远[2] SUN Haipeng;XU Lufan;LIU Xue;LIU Siyuan(Affiliated Hospital of Shandong University of Traditional Chinese Medicine(TCM),Jinan 250014,China;Shandong University of TCM,Jinan 250355,China)

机构地区：[1]山东中医药大学附属医院,济南250014 [2]山东中医药大学,济南250355

出　　处：《中国实验方剂学杂志》2025年第4期116-124,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金青年基金项目(82004285);山东省自然科学基金青年项目(ZR2023QH072);山东省中医药科技项目(2021Q076);山东中医药大学中医传统功法康复青年科研探索团队。

摘　　要：目的:从细胞水平探讨特异性蛋白1(Sp1)-微RNA-582-3p(miR-582-3p)-细胞周期蛋白依赖性激酶抑制剂(p27)轴过度激活对A549肺腺癌细胞周期、增殖影响,以研究黄芪-白花蛇舌草可能调控该轴抑制A549增殖的相关机制。方法:采用慢病毒质粒转染技术,获得sh RNA-Sp1+LV-miR-582-3p mimic、sh RNA-Sp1+LV-m NC、sh RNA-NC+LV-miR-582-3p mimic、sh RNA-NC+LV-m NC的A5494组稳转株。实时荧光定量聚合酶链式反应(Real-time PCR)检测Sp1对A549肺腺癌细胞miR-582-3p表达影响;细胞增殖与活性检测法(CCK-8)、平板克隆形成实验检测Sp1通过miR-582-3p对A549肺腺癌细胞增殖影响;流式细胞术检测Sp1通过miR-582-3p对A549肺腺癌细胞周期影响;蛋白免疫印迹法(Western blot)检测Sp1通过miR-582-3p对A549肺腺癌细胞周期蛋白的影响。CCK-8检测5%、10%、20%、40%、80%黄芪-白花蛇舌草含药血清对A549细胞活力影响,并筛选出最佳浓度用于后续机制探究。上述转染技术构建oe-Sp1稳转株及对照株oe-NC,Real-time PCR检测黄芪-白花蛇舌草对A549肺腺癌细胞miR-582-3p表达影响;Western blot检测黄芪-白花蛇舌草对A549肺腺癌细胞Sp1、细胞周期依赖性激酶特异性抑制蛋白p27表达影响。结果:与sh RNA-NC+LV-m NC组比较,sh RNA-Sp1+LV-m NC组miR-582-3p表达显著降低(P<0.01);与sh RNA-Sp1+LV-m NC组比较,sh RNA-Sp1+LV-miR-582-3p mimic组显著逆转miR-582-3p表达(P<0.01),提示Sp1对miR-582-3p具有调控作用。与sh RNA-Sp1+LV-m NC组比较,sh RNA-Sp1+LV-miR-582-3p mimic组显著逆转细胞增殖(P<0.01),提示miR-582-3p可部分逆转Sp1对A549肺腺癌增殖作用;与sh RNA-Sp1+LV-m NC组比较,sh RNA-Sp1+LV-miR-582-3p mimic组G_(0)/G_(1)期比例显著降低,S期、G_(2)/M期比例显著升高(P<0.01),提示miR-582-3p可部分逆转Sp1对A549细胞周期影响;与sh RNA-Sp1+LV-m NC组比较,sh RNA-Sp1+LV-miR-582-3p mimic组显著逆转各蛋白水平,即p27的下降与细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白依赖激�Objective:To investigate the effects of excessive activation of the Specific protein 1(Sp1)-Micro RNA-582-3p(miR-582-3p)-cyclin-dependent kinase inhibitor(p27)axis on the cell cycle and proliferation of A549 lung adenocarcinoma cells at the cellular level,thereby investigating the possible mechanisms by which Astragali Radix and Hedyotis diffusa(A-H)regulate the axis to inhibit A549 cells proliferation.Methods:Lentiviral plasmid transfection technology was used to obtain four stable A549transgenic cell lines:sh RNA-Sp1+LV-miR-582-3p mimic,sh RNA-Sp1+LV-m NC,sh RNA-NC+LV-miR-582-3p mimic,and sh RNA-NC+LV-m NC.Real-time PCR was used to detect the effect of Sp1 on miR-582-3p expression in A549 lung adenocarcinoma cells.CCK-8 and colony formation assay were used to detect the effect of Sp1 on cell proliferation through miR-582-3p,while flow cytometry was used to detect the effect of Sp1 on cell cycle via miR-582-3p.Western blot was used to detect the effect of Sp1 on cyclin in A549 cells through miR-582-3p.CCK-8 was employed to detect the effects of different concentrations(5%,10%,20%,40%,80%)of A-H on A549 cell viability,and the optimal concentration was selected for subsequent mechanism exploration.Stable transgenic strains oe-Sp1 and control oe-Vector were constructed using the above-mentioned transfection techniques.Realtime PCR was then used to investigate the effect of A-H on miR-582-3p expression in A549 cells,while Western blot was employed to detect the effect of A-H on Sp1 and p27 expression in A549 lung adenocarcinoma cells.Results:Compared with the sh RNA-NC+LV-m NC group,the expression of miR-582-3p was significantly inhibited in the sh RNA-Sp1+LV-m NC group(P<0.01).Compared with the sh RNA-Sp1+LV-m NC group,the sh RNA-Sp1+LV-miR-582-3p mimic group significantly reversed miR-582-3p expression(P<0.01),indicating that Sp1 has a significant regulatory effect on miR-582-3p.Compared with the sh RNA-Sp1+LV-m NC group,the sh RNA-Sp1+LV-miR-582-3p mimic group significantly reversed cell proliferation(P<0.01),sug

关 键 词：黄芪-白花蛇舌草 Sp1-miR-582-3p-p27轴 A549肺腺癌细胞 周期 增殖 

分 类 号：R256[医药卫生—中医内科学] R734.2[医药卫生—中医学] R285.5