基础医学与临床 ›› 2022, Vol. 42 ›› Issue (1): 106-113.

• 研究论文 • 上一篇    下一篇

eEF2K沉默联合丹参酮ⅡA磺酸钠协同抑制人肺腺癌细胞系A549增殖

王布,袁胜芳,张长洪,苑程,黄攀登,张志华,赵建清   

  1. 河北北方学院附属第一医院
  • 收稿日期:2020-11-09 修回日期:2021-04-12 出版日期:2022-01-05 发布日期:2022-01-05
  • 通讯作者: 赵建清 E-mail:zauu62@163.com
  • 基金资助:
    河北省卫生厅科研基金项目;张家口市科技攻关计划项目

Silencing eEF2K combined with sodium tanshinone IIA sulfonate synergistically inhibit proliferation of human lung adenocarcinoma cell line A549

  • Received:2020-11-09 Revised:2021-04-12 Online:2022-01-05 Published:2022-01-05

摘要: 目的:探讨真核延伸因子激酶-2(eEF-2K)基因转染及丹参酮ⅡA磺酸钠(STS)对肺腺癌A549细胞增殖、侵袭和迁移的影响及其机制。方法:以0、1.25、2.5、5、10和20 μg/mL STS作用48 h后,采用细胞计数试剂盒-8(CCK-8)法检测A549细胞存活率以筛选STS作用浓度。将靶向eEF-2K基因的siRNA(siRNA-eEF-2K)转染至A549细胞后,采用免疫印迹法和实时荧光定量PCR分别检测A549细胞中eEF-2K蛋白和mRNA表达水平。将体外培养的A549细胞分为siRNA-NC组(转染siRNA-NC)、siRNA-eEF-2K组(转染siRNA-eEF-2K)、siRNA-NC+STS组(转染siRNA-NC后给予10 μg/mL STS)和siRNA-eEF-2K+STS组(转染siRNA-eEF-2K后给予10 μg/mL STS),采用CCK-8法、克隆形成实验、Transwell小室实验、划痕实验和流式细胞仪分别检测各组A549细胞存活率、克隆形成率、穿膜细胞数、迁移率和凋亡率,免疫印迹法检测各组A549细胞中蛋白激酶B(AKT)、磷酸化(p)-AKT、Ki67、基质金属蛋白酶2(MMP-2)和B淋巴细胞瘤-2基因(Bcl-2)蛋白表达水平。结果:与0 μg/mL比较,1.25、2.5、5、10和20 μg/mL STS作用后A549细胞存活率均明显降低(P<0.05),且呈一定的浓度依赖性;STS对A549细胞的半数抑制浓度为10.39 μg/mL。转染siRNA-eEF-2K可成功抑制A549细胞中eEF-2K蛋白和mRNA表达。与siRNA-NC组比较,siRNA-eEF-2K组、siRNA-NC+STS组和siRNA-eEF-2K+STS组细胞存活率、克隆形成率、穿膜细胞数、迁移率和细胞中p-AKT、Ki67、MMP-2、Bcl-2蛋白均明显降低,而细胞凋亡率明显升高(P<0.05),且siRNA-eEF-2K+STS组中上述指标变化幅度明显大于siRNA-NC+STS组和siRNA-eEF-2K组。结论:eEF-2K基因沉默联合STS可协同抑制A549细胞增殖、侵袭和迁移并促进细胞凋亡,其作用机制可能与共同抑制p-AKT、Ki67、MMP-2、Bcl-2蛋白表达有关。

关键词: 肺腺癌, 真核延伸因子激酶-2, 丹参酮ⅡA磺酸钠, 细胞增殖, 细胞侵袭, 细胞凋亡

Abstract: Objective: To investigate the effects of eukaryotic elongation factor 2 kinase (eEF-2K) gene transfection and sodium tanshinone IIA sulfonate (STS) on proliferation, invasion and migration of lung adenocarcinoma A549 cells and its mechanism. Methods: After treated with 0, 1.25, 2.5, 5, 10 and 20 μg/mL STS for 48 h, the survival rate of A549 cells was detected by cell counting kit-8 (CCK-8) method to screen the concentration of STS. After siRNA targeting eEF-2K gene (siRNA-eEF-2K) was transfected into A549 cells, the expression levels of eEF-2K protein and mRNA in A549 cells were detected by Western blot and real-time quantitative PCR. A549 cells were divided into siRNA-NC group (transfected with siRNA-NC), siRNA-eEF-2K group (transfected with siRNA-eEF-2K), siRNA-NC + STS group (treated with 10 μg/mL STS after siRNA-NC) and siRNA-eEF-2K + STS group (treated with 10 μg/mL STS after siRNA-eEF-2K), the survival rate, clone formation rate, number of transmembrane cells, migration rate and apoptosis rate of A549 cells were detected by CCK-8 method, clone formation test, Transwell chamber test, scratch test and flow cytometry, the protein expression levels of protein kinase B (AKT), phosphorylation (p)-AKT, Ki67, matrix metalloproteinase-2 (MMP-2) and B-lymphoma-2 genes (Bcl-2) were detected by Western blot. Results: Compared with 0 μg/mL STS, the survival rate of A549 cells was significantly decreased after treatment with 1.25, 2.5, 5, 10 and 20 μg/mL STS (P < 0.05), and it was in a concentration dependent manner; the 50% inhibitory concentration of STS on A549 cells was 10.39 μg/mL. Transfection of siRNA-eEF-2K can successfully inhibit the expression of eEF-2K protein and mRNA in A549 cells. Compared with those in siRNA-NC group, the cell survival rate, clone formation rate, number of transmembrane cells, migration rate and p-AKT, Ki67, MMP-2 and Bcl-2 in proteins siRNA-eEF-2K + STS group were significantly lower, while the apoptosis rate was significantly higher (P < 0.05), the changes of the above indexes in siRNA-eEF-2K + STS group were significantly greater than those in siRNA-NC + STS group and siRNA-eEF-2K group. Conclusions: The eEF-2K gene silencing combined with STS can synergistically inhibit the proliferation, invasion and migration of A549 cells and promote cell apoptosis. The mechanism may be related to the inhibition of protein expression of p-AKT, Ki67, MMP-2 and Bcl-2.

Key words: lung adenocarcinoma, eukaryotic elongation factor 2 kinase, sodium tanshinone IIA sulfonate, cell proliferation, cell invasion, apoptosis