RNA干扰沉默NS基因对人非小细胞肺癌A549细胞增殖和凋亡的影响

Effect of RNAi-mediated silencing of nucleostemin gene on proliferation and apoptosis of human non-small cell lung cancer A549 cells

目的:研究RNA干扰沉默核干细胞因子(nucleostemin,NS)基因表达对人肺腺癌A549细胞株增殖和凋亡的影响。方法:向A549细胞内分别转染靶向NS基因的siRNA表达载体pcDNA4/C-NS-silencer和空载体pcDNA4/C vector作为silencer组和vector组,以不转染质粒的A549细胞为normal组,Real-time PCR检测转染pcDNA4/C-NS-silencer对A549细胞内NS基因表达的影响。CCK-8法检测沉默NS基因对A549细胞增殖的影响,流式细胞术检测对细胞周期的影响,Hoechst33258核染色法和流式细胞术检测对细胞凋亡的影响。结果:Silencer组NS基因表达较vector组和normal组明显被抑制(0.166±0.024 vs 0.497±0.022、0.505±0.032,均P<0.01);Silencer组A549细胞增殖活性显著低于vector组和normal组(0.518±0.107 vs 0.855±0.102、0.832±0.158,均P<0.05);Silencer组A549细胞周期阻滞于G0/G1期;Silencer组细胞核皱缩呈致密浓染,染色质碎裂呈块状并有边集现象,且细胞凋亡率较vector组与normal组显著增高[(34.80±6.77)%vs(9.70±1.50)%,(8.16±2.01)%,P<0.01]。结论:RNA干扰沉默NS基因可抑制人肺腺癌A549细胞的增殖,促进细胞凋亡。

Objective： To determine the effect of silencing nucleostemin（ NS） gene expression on proliferation and apoptosis of non-small cell lung cancer（ NSCLC） A549 cells in vitro. Methods： The recombinant pcDNA4 /C-NS-silencer targeting the NS gene was constructed. Human NSCLS A549 cells were transfected with pcDNA4 /C-NS-silencer or the control vectorpcDNA4 /C. NS mRNA abundance was assessed by real-time RT-PCR,cell proliferation by CCK-8 assay,and cell cycle progress and apoptosis by flow cytometric assay with Hoechst33258 staining in untransfected and transfected A549 cells. Results： Compared with control vector-transfected and untransfected A549 cells,A549 cells transfected with pcDNA4 /C-NS-silencer had significantly lower NS mRNA abundance（ 0. 166 ± 0. 024 vs 0. 497 ± 0. 022,0. 505 ± 0. 032,P〈 0. 01） and a significantly lower proliferation activity（ 0. 518 ± 0. 107 vs 0. 855 ± 0. 102,0. 832 ± 0. 158,P 〈0. 05）.NS gene silencing resulted significant increases in the percentage of cells in G1/G0 phase,in chromatin condensation andfragmentation,and in apoptosis（ [34. 80 ± 6. 77]% vs [9. 70 ± 1. 50]%,[8. 16 ± 2. 01]% P〈 0. 01）,as compared with control vector-transfected and untransfected A549 cells. Conclusion： RNAi-mediated silencing of the NS gene results in human non-small cell lung cancer cell proliferation,G1 arrest and apoptosis,thereby possessing a therapeutic potential.