斯钙素1的表达对肺癌细胞A549细胞周期及凋亡的影响

Effects of stanniocalcin l on cell cycle and apoptosis of lung cancer A549 cells

背景与目的:斯钙素1(stanniocalcin l,STC1)在多种癌组织中表达上调,且与癌组织的恶性程度相关,但STC1在肺癌细胞中的分子作用机制尚不明确。本研究旨在探讨STC1的表达对肺癌细胞A549细胞周期及凋亡的影响。方法:构建STC1基因RNA干扰的肺癌细胞株A549-STC1-si RNA和对照细胞株A549-Vector,用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测A549-Vector及A549-STC1-si RNA细胞株的细胞周期蛋白基因Cyclin A、Cyclin B1、Cyclin D1、Cyclin E、CDK2、CDK4,凋亡抑制基因Bcl-2、Bcl-xl及凋亡诱导基因Caspase-3、Bax、Bak、Bid的表达水平,用流式细胞术检测STC1基因对A549细胞周期的影响,用原位末端标记(terminal deoxynucleotidyl transferase-mediated nick-end labeling,TUNEL)检测STC1基因对A549细胞凋亡的影响。结果:与A549-Vector细胞相比,A549-STC1-si RNA的细胞周期蛋白基因Cyclin A、Cyclin B1、Cyclin D1、Cyclin E、CDK2和CDK4在转录和蛋白表达水平上均显著减少(P<0.05),G_0/G_1期细胞比例明显增加,S期及G_2/M期细胞比例降低(P<0.05),细胞周期受阻;A549-STC1-si RNA的凋亡抑制基因Bcl-2和Bcl-xl表达下调(P<0.05),而凋亡诱导基因Caspase-3、Bax、Bak及Bid显著上调(P<0.05);TUNEL实验表明,A549-STC1-si RNA细胞的凋亡率明显增加。结论:STC1基因的低表达可阻滞肺癌细胞A549的细胞周期,抑制细胞增殖,同时促进细胞凋亡。

Background and purpose： Stanniocalcin 1 （STC1） has been reported to be up-regulated in various cancer tissues, and related to malignancy degree of cancer. However, the molecular mechanism of STC 1 in lung cancer cells is still not dear. This experiment aimed to investigate the effects of STC1 on cell cycle and apoptosis of lung cancer A549 cells. Methods： A549 cells were transfected with validated siRNA for STC1 A549-STC1-siRNA and a negative control vector RNA A549-Vector. The gen~ and protein expression of cell cycle-related genes, including CyclinA, CyclinB1, CyclinD1, CyclinE, CDK2 and CDK4, as well as apoptosis-inhibiting genes Bcl-2, Bcl-xl and apoptosis-inducing genes Caspase-3, Bax, Bak and Bid, were detected by real-time fluorescent quantitative polymerase chain reaction （RTFQ-PCR） and Western blot. The cell cycle distribution was determined with flow cytometry. Terminal deoxynucleotidyl transferase-mediated nick-end labeling （TUNEL） was used to detect cell apoptosis. Results： After transfection with STCI-siRNA, the gene and protein expression of CyclinA, CyclinB1, CyclinD1, CyclinE, CDK2 and CDK4 decreased significantly in A549 cells （P〈0.05）. The proportion of cells in Go/Gt phase significantly increased,whereas the proportion of cells in S phase and G2/M phase decreased （P〈0.05）. The cell cycle was blocked at G0/G1 phase. Furthermore, compared with that in A549-Vector, the gene and protein expression of Bc1-2 and Bcl-xl in A549- STC1-siRNA was reduced significantly （P〈0.05）, while the expression of apoptosis-inducing genes Caspase-3, Bax, Bak and Bid increased obviously （P〈0.05）. In addition, the percentage of apoptotic cells significantly increased in A549-STCl-siRNA compared with that in A549-Vector detected by TUNEL method. Conclusion： Down-regulation of STC1 by RNAi can block the cell cycle ofA549 cells, inhibit cell proliferation, and promote cell apoptosis.