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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 target retroviral vector targeting GW112 siRNA and screen specific and efficient RNAi target to study the effect of GW112 gene silencing on the proliferation of gastric cancer cell line SGC-7901 in vitro. Methods RNA interference vector-pSiRNA-GW112 (pSi405-GW112, pSi1066-GW112, pSi1480-GW112) targeting to three different targets of GW112 gene was constructed by retroviral vector psilencer5.1-H1Retro. Liposome was transfected into PT67 The cell-packaged virus was used to infect SGC-7901 cells. Puromycin was used to screen the stable clones. RT-PCR was used to identify the virus integration. Real-time PCR screening was used to efficiently inhibit the target site. CCK-8 assay was used to detect the proliferation of SGC-7901 cells. Western blot was used to detect the expression of proliferating cell nuclear antigen (PCNA). Results The sequencing results confirmed that the recombinant retroviral vector pSiRNA-GW112 was constructed correctly. The results of RT-PCR showed that all the cells infected with target virus supernatant could amplify 510 bp Puro resistance gene except for the parental cells. Real-Time PCR analysis showed that the GW112 transcription level of the 7901-Si405, 7901-Si1066 and 7901-Si1480 groups was (15.50 ± 0.01)% and (32.40 ± 0.01) times of the SGC-7901 group % And (57.00 ± 0.02)%. However, the expression of GW112 in 7901-Sc group was almost unchanged. The inhibitory rates of Si405, Si1066 and Si1480 were 84.5%, 67.6% and 43.0%, respectively. Silencing GW112 resulted in significant inhibition of proliferation of SGC-7901 cells in vitro (P <0.05), accompanied by down-regulation of PCNA protein expression. Conclusion The recombinant retroviral vector pSiRNA-GW112 can significantly inhibit the proliferation of SGC-7901 cells in vitro, and this inhibition of proliferation is related to the down-regulation of PCNA expression.