RNA干扰技术抑制smad4基因表达对宫颈癌细胞增殖的影响

目的：探讨用RNA干扰技术下调smad4基因表达对宫颈癌细胞增殖的影响。方法：用DNA重组技术将针对人smad4基因不同部位所设计的三对短发夹状RNA（shorthairpinRNA，shRNA）序列克隆到真核表达质粒pGenesil-1中，构建smad4shRNA表达载体pGenesil—smad4一shRNA1、2、3。脂质体介导转染人宫颈癌细胞株HeLa，经G418筛选抑制smad4表达的稳定细胞克隆。MTT法、克隆形成实验、流式细胞术检测抑制smad4基因对宫颈癌细胞增殖的影响。结果：成功构建分别携带三段shRNA及空载体对照的重组质粒pGenesil—smad4-shRNA1、2、3和pGenesil-con，三种shRNA重组质粒中pGenesil—smad4-shRNA2可明显降低细胞内smad4mRNA及smad4蛋白表达，筛选出的HeLa／shRNA2细胞增殖能力明显增强。结论：应用RNA干扰技术能筛选出特异而高效阻断smad4基因表达及功能的shRNA，smad4基因表达下调能明显促进宫颈癌细胞生长。

Influence of RNA interference-induced smad 4 gene silencing on the proliferation of cervical cancer cells

Objective:To explore the feasibility of selective inhibiting smad 4 expression by using smad4 short hairpin RNA (shRNA)interference,and observe the effects of smad 4 gene silencing on HeLa cells growth.Methods:Three 19-bp reverse re peated motifs(shRNA)targeting three different domains of smad 4 gene were designed,synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase to construct recombi- nant plasmids pGenesil-smad4-shRNA1,2,and 3.The three recombinant plasmids and pGenesil-con were transfected into HeLa ceils via lipofectamine mediation.The stable clone was screened by G418.The alteration of smad4 expression was examined by real-time polymerase chain reaction and Western blot.The proliferation capacity of HeLa cells was measured by MTT assay, colony formation assay,and cell cycles analysis.Results:Recombinant plasmids containing three shRNA(pGenesil smad4-shR- NA1,2,and 3 and control plasmid(pGenesil-con)were successfully constructed and transfected into HeLa cells,respectively. Transfection of pGenesil-smad4 shRNA2 significantly knocked down the smad4 mRNA and protein expression.The proliferation capacity of HeLa cells transfected with pGenesil-shRNA2 was significantly enhanced compared with those transfected with pGen- esil-con(H/con).Conelusion:The specific smad4 targeted shRNA can efficiently suppress smad4 expression in HeLa cells.Se- lective smad 4 gene silencing can stimulate the proliferation of cervical cancer cells.