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  

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

作　　者：闫静川 余彦平 乔一桓 黄硕 丁晓琛 刘俊 姜勋亮 王贺 李纪鹏 YAN Jing-chuan;YU Yan-ping;QIAO Yi-huan;HUANG Shuo;DING Xiao-chen;LIU Jun;JIANG Xun-liang;WANG He;LI Ji-peng(School of Basic Medicine,Air Force Military Medical University,Xi'an,Shaanxi,710032,China;Shaanxi Provincial Tumor Hospital,Xi'an,Shaanxi,710061,China;School of Clinical Medicine,Shaanxi,Xi'an Medical University,Xi'an,Shaanxi,710021,China;Experimental surgery,Xijing Hospital,Air Force Military Medical University,Xi'an,Shaanxi,710032,China;State Key Laboratory of Cancer Biology,Department of Biochemistry and Molecular Biology,Air Force Military Medical University,Xi'an,Shaanxi,710032,China;Department of digestive surgery,Xijing Hospital,Air Force Military Medical University,Xi'an,Shaanxi,710032,China)

机构地区：[1]空军军医大学基础医学院,陕西西安710032 [2]陕西省肿瘤医院,陕西西安710061 [3]西安医学院临床医学院,陕西西安710021 [4]空军军医大学第一附属医院西京医院实验外科,陕西西安710032 [5]空军军医大学生物化学与分子生物学教研室,肿瘤生物学国家重点实验室,陕西西安710032 [6]空军军医大学第一附属医院西京医院消化病院消化外科,陕西西安710032

出　　处：《现代生物医学进展》2020年第17期3212-3216,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81672751);陕西省重点研发计划项目(2019SF-010)。

摘　　要：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-qPCR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(P<0.001);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(P<0.001),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(P<0.01)。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。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 HEK293 T 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 5 th 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). Conclusions: 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.

关 键 词：核仁素 慢病毒 基因沉默 胃癌 增殖 

分 类 号：R-33[医药卫生] Q78[生物学—分子生物学]