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| 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* |
| 投稿时间：2020-01-02 修订日期：2020-02-25 |
| DOI： |
| 中文关键词: 核仁素 慢病毒 基因沉默 胃癌 增殖 |
| 英文关键词: necleolin lentivirus gene silencing gastric cancer proliferation |
| 基金项目:国家自然科学基金资助项目(No. 81672751)；陕西省重点研发计划(No. 2019SF-010)。 |
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| 中文摘要: |
| 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：成功建立了慢病毒介导的NCL沉默的胃癌细胞系SGC-7901,并且揭示了沉默NCL基因能够抑制SGC-7901细胞的增殖能力的观点,为理解胃癌发生发展的分子机理提供新的理论基础。 |
| 英文摘要: |
| 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 gastric cancer. Method(s): Using small hairpin RNA (shRNA) mediated lentivirus system to silence NCL in gastric cancer cells. 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. Result(s): Agarose gel electrophoresis was used to detect the pKLO.1-NCL vector identified by restriction enzyme digestion, showing two bands located at 5000 bp and 2000 bp. The sequence map showed the same sequence as the designed. Lentivirus was produced using HEK293T cells, and then infects 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 was 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 was significantly reduce, and the number of clones were dropped significantly (P < 0.01). Conclusion(s): We establish a lentivirus mediated NCL silenced in gastric cancer cell line SGC-7901 successfully, and we demonstrate that silencing NCL gene can inhibit the proliferation of SGC-7901 cells. Our finding can provide a new theoretical basis for making a better understanding of the molecular mechanism of gastric cancer. |
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