慢病毒介导NCL基因沉默的胃癌细胞系的建立及对细胞增殖影响的研究

目的:本研究通过建立慢病毒介导的NCL基因沉默的胃癌细胞系,研究NCL沉默对胃癌细胞增殖能力的影响,为深入探究胃癌发生发展的分子机制提供理论基础。方法:利用小发卡RNA(shRNA)介导的慢病毒系统沉默胃癌细胞中的NCL,并利用RT-q PCR和免疫印迹检测基因沉默效果;并利用CCK-8实验和平板克隆形成实验检测胃癌细胞的增殖能力的改变。结果:琼脂糖凝胶电泳实验检测经酶切鉴定的pKLO.1-NCL载体,显示5000 bp和2000 bp两条带,测序峰图显示与设计序列一致;利用HEK293T包装病毒,感染胃癌细胞SGC-7901,免疫印迹结果显示sh NCL组NCL蛋白水平显著低于对照组,RT-qPCR结果显示,sh NCL组NCL表达量显著降低,为对照组的0.4209±0.087倍(P0.001);CCK-8实验结果显示,sh NCL组在第5天的吸光值较对照组显著降低(P0.001),平板克隆形成实验结果显示,sh NCL组克隆形成能力较对照组显著降低,克隆形成数量显著低于对照组(P0.01)。结论:建立了慢病毒介导的NCL基因沉默的胃癌细胞系SGC-7901,并利用此系统研究了NCL基因对胃癌细胞增殖能力的影响,证明了NCL基因能够促进胃癌细胞的增殖,为后续研究NCL基因在胃癌细胞中的作用提供基础。

Establishment of a Lentivirus Mediated NCL Gene Silencing System and Its Effect on Proliferation of Gastric Cancer Cells

ABSTRACT Objective: In this study, we established a gastric cancer cell line with lentivirus mediated NCL gene silencing, and studied the effect of NCL silencing on the proliferation of gastric cancer cells. We provide a theoretical basis for further exploring the molecular mechanism of NCL in gastric cancer. Methods: Small hairpin RNA (shRNA) mediated lentivirus system was used to silence NCL gene in gastric cancer cells. And RT-qPCR and immunoblotting were used to detect gene silencing effect. CCK-8 assay and plate clone formation experiment were used to detect the proliferation of gastric cancer cells. Results: Agarose gel electrophoresis was used to detect the pKLO.1-NCL vector identified by restriction enzyme digestion, and the result showed two bands located at 5000 bp and 2000 bp. Lentivirus was produced by using HEK293T cells, and then infected SGC-7901 gastric cancer cells. Western blot showed that the protein level of NCL in sh NCL group decreases significantly compared with that in control group. RT-qPCR showed that the expression of NCL in sh NCL group reduces significantly compared with that in control group, which was 0.4209 ± 0.087 times than that in control group (P<0.001). CCK-8 assay showed that the absorbance value of sh NCL group on the 5th day significantly reduces compared with that of control group (P<0.001). The plate clone formation experiment showed that the clone formation ability of sh NCL group significantly reduces, and the number of clones drops significantly (P<0.01). Conclusion: We establish a lentivirus mediated NCL silenced system in gastric cancer cell line SGC-7901 successfully, and demonstrate the silencing of NCL gene in SGC-7901 cells can inhibit the proliferation of cells. Our finding can provide a basis for the study of the role of NCL gene in gastric cancer cells.