GW112基因siRNA对人胃癌SGC-7901细胞体外增殖的影响

Inhibitory effect of silencing GW112 siRNA gene on proliferation of human gastric SGC-7901 cell line

目的构建靶向GW112siRNA逆转录病毒载体并筛选特异高效的RNAi靶点,研究GW112基因沉默后对胃癌SGC-7901细胞体外增殖的影响。方法应用逆转录病毒载体psilencer5.1-H1Retro构建针对GW112基因3个不同靶点的RNAi干扰载体-pSiRNA-GW112(pSi405-GW112,pSi1066-GW112,pSi1480-GW112),将脂质体法转染PT67细胞包装的病毒感染SGC-7901细胞,嘌呤霉素筛选稳定克隆,RT-PCR鉴定病毒整合,Real-timePCR筛选高效抑制靶点,CCK-8法检测对细胞体外增殖的影响。Westernblot检测增殖细胞核抗原(PCNA)的表达。结果测序结果证实各干扰靶点的重组逆转录病毒载体pSiRNA-GW112构建正确。RT-PCR结果显示除亲本细胞外,经各靶点病毒上清感染的细胞均能扩增出510bp的Puro抗性基因片段。Real-TimePCR分析表明与SGC-7901亲本细胞组相比,7901-Si405,7901-Si1066及7901-Si1480组GW112的转录水平分别只有SGC-7901组的(15.50±0.01)%,(32.40±0.01)%与(57.00±0.02)%。而7901-Sc组GW112表达基本无变化。Si405,Si1066与Si1480靶点的抑制率分别为84.5%,67.6%及43.0%。沉默GW112后导致SGC-7901细胞体外增殖能力显著抑制(P<0.05),并伴随PCNA蛋白表达下调。结论构建的重组逆转录病毒载体pSiRNA-GW112能显著抑制SGC-7901细胞的体外增殖能力,且这种增殖抑制与下调的PCNA表达有关。

Objective To construct the small interfering RNA （siRNA） retrovirus vector targeting GW112 with a high specificity and to study the effect of silencing GW112 gene on the proliferation of gastric SGC-7901 cells.Methods Three recombinant RNAi interference vectors were constructed according to different GW112 targeting points （pSi405-GW112,pSi1066-GW112,pSi1480-GW112） using a retrovirus expression vector,psilencer 5.1-H1 Retro.SGC-7901 cells were infected with a packaged PT67 cells transfected with liposome.Stable clones were selected using puromycin.Integration of virus was identified by RT-PCR and highly inhibited targets were detected RT-PCR.The effect of GW112 on proliferation of gastric SGC-7901 cells was detected with CCK-8 kit.Expression of proliferating cell nuclear antigen （PCNA） was assayed by Western blotting.Results Sequence analysis showed recombinant retrovirus vector psiRNA-GW112 was successfully constructed.RT-PCR showed that except for parental cells,other SGC-7901 cells infected with virus supernatant could amplify anti-puromycin GW112 gene into a 510 bp fragment.The inhibitory efficacy was 84.5%,67.6%,and 43.0%,respectively,for Si405,Si1066 and Si1480 respectively.Both the proliferation of gastric SGC-7901 cells and the expression of PCNA in SGC-7901 cells were significantly inhibited after GW112 was silenced.Conclusion The recombinant retrovirus vector,pSiRNA-GW112,can significantly inhibit the proliferation and expression of PCNA in SGC-7901 cells,indicating that GW112 plays an important role in proliferation of SGC-7901cells.