STAT3siRNA对肺腺癌A549细胞STAT3表达和细胞生物学行为的影响

Silencing of STAT3 by STAT3siRNA influences biological behavior of lung adenocarcinoma cell line A549

目的:观察经RNA干扰沉默STAT3基因对人肺腺癌细胞A549生长和凋亡的影响。方法:设计并经化学合成了针对STAT3基因不同位点的3条小干扰RNA(small interfering RNA,siRNA)序列(STAT3siRNA1、STAT3siRNA2、STAT3siRNA3),在脂质体(LipofectamineTM2000)介导下转染人肺腺癌细胞株A549,采用RT-PCR法检测STAT3mRNA表达变化,筛选出抑制作用最强的STAT3-siRNA。免疫印迹法检测细胞STAT3、Cyclin D1、BAX、Bcl-2、Caspase-9蛋白表达。AM-BLUE法测定A549细胞体外增殖情况。流式细胞仪检测细胞凋亡。结果:经RNA干扰能有效降低A549细胞STAT3mRNA表达水平(P<0.05),转染STAT3siRNA后,A549细胞STAT3、Cyclin D1、Bcl-2蛋白的表达水平均显著降低(P<0.05),而BAX、Caspase-9蛋白的表达水平升高(P<0.05)。STAT3蛋白表达分别与CyclinD1、Bcl-2蛋白表达呈正相关(r=0.854,0.910,P<0.05);与BAX蛋白表达呈负相关性(r=-0.848,P<0.05);与Caspase-9蛋白表达呈无相关性(r=-0.787,P>0.05)。RNA干扰法沉默STAT3基因表达后,A549细胞增殖受到明显抑制,细胞凋亡率明显增加。结论:STAT3siRNA可有效抑制A549细胞STAT3表达,抑制A549细胞生长并促进其凋亡。

Objective：Effects of STAT3-siRNA-mediated gene silencing on bionomics and growth inhibition in the lung adenocarcinoma cell line A549 was investigated.Methods：Based on the sequence of mRNA,three siRNAs were designed and chemically synthesized.They were transfected into the lung adenocarcinoma cell line A549 with LipofectamineTM 2000,expression of STAT3 mRNA and protein levels was determined by RT-PCR and Western blot.Most powerful inhibition of STAT3-siRNA for expression of STAT was selected.Cell growth inhibition and apoptosis were determined by AM-BLUE assay and flow cytometry.Results：Short siRNAs targeting STAT3 down-regulated expression of STAT3 and STAT3,CyclinD1,Bcl-2 in mRNA and protein levels（P〈0.05）,and up-regulated expression of BAX,Caspase-9 protein（P〈0.05）.The expression of STAT3 protein in A549 cell treated with siRNA was positively correlated with CyclinD1,Bcl-2（r=0.854,0.910,P〈0.05）,and was negatively correlated with BAX（r=-0.848,P〈0.05）.However,no correlation was found between STAT3 and Caspase-9（r=-0.787,P〉0.05）.The proliferation of A549 cells was inhibited（P〈0.05）and the ratio of apoptosis significantly increased after transfection（P〈0.05）.Conclusion：STAT3-siRNAs can significantly down-regulate STAT3 expression in transcriptional and translational levels,inhibit cell growth and induce cell apoptosis in the lung adenocarcinoma cell line A549.