The Effect of Silencing of UBE2C Gene on the Proliferation and Migration of Human Gastric Cancer AGS Cells
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摘要:
目的 探讨UBE2C基因沉默对人胃癌AGS细胞增殖和迁移能力的影响。 方法 将用RNA 干扰技术构建的UBE2C-shRNA表达载体转染到293T细胞，用含有病毒颗粒的上清感染人胃癌AGS细胞，经puromycin筛选获得稳定表达的AGS细胞株。实验分为UBE2C表达沉默人胃癌AGS细胞组（干扰组）和对照组。采用qRT-PCR验证人胃癌AGS细胞中UBE2C的mRNA 表达水平，随后利用实时无标记细胞分析仪（RTCA）检测UBE2C沉默对人胃癌AGS细胞的增殖和迁移情况；同时应用CCK-8 细胞增殖检测试剂盒进一步印证UBE2C表达水平对人胃癌AGS细胞增殖能力影响。 结果 干扰组中UBE2C mRNA 表达 水平与对照组相比明显降低，AGS细胞增殖和迁移能力显著下降（P < 0.01）。 结论 靶向沉默UBE2C基因表达能抑制人胃癌AGS细胞恶性生物学行为。 Abstract:Objective To investigate the influence of the silence of UBE2C gene on the gastric cancer AGS cells proliferation and migration of human. Methods Using RNA interference technology, the expression vector of UBE2C silenced lentivirus was transfered into 293T cell line with packaging plasmids to produce the lentiviral particles. Lentivirus was used to infect human gastric cancer AGS cells, and the cells with stably expressing UBE2C were obtained by puromycin screening. The experiment was divided into the interference group and the interference control group. The expression levels of UBE2C mRNA in human gastric cancer AGS cells were detected by quantitative real-time polymerase chain reaction （qRT-PCR）. The capacity of AGS cell proliferation and migration was determined with real-time label free cell analyzer （RTCA）. Meanwhile, Cell counting kit-8 （CCK-8） was used to further confirm the effect of UBE2C expression level on the proliferation of human gastric cancer AGS cells. Results Compared with the interference control group, UBE2C shRNA transfected human gastric cancer AGS cells significantly decreased the expression of UBE2C mRNA （P < 0.05）; the cellular proliferation and migration ability were obviously decreased. Conclusion Targeted silencing of UBE2C gene expression can inhibit the malignant biological behavior of human gastric cancer AGS cells. -
Key words:
- UBE2C /
- human gastric cancer AGS cells /
- proliferation /
- Migration
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图 1 293T细胞转染3组慢病毒干扰质粒和对照质粒72 h荧光显微镜观察结果（100×）
Figure 1. 293 T cells transfected with three group of interfering lentiviral vectors and control vector for 72hours were photographed under fluorescene microscope（100×）
A：Contrl-shRNA；B：UBE2C -shRNA1；C：UBE2C -shRNA2；D：UBE2C -shRNA3。
图 2 UBE2C-shRNA转染对UBE2C表达的影响（*P < 0.05，与对照组相比，n = 3）
与Control-shRNA组比较，*P < 0.05，n = 3。
Figure 2. Interference efficiency of UBE2C-shRNA plasmid on UBE2C expression
图 3 荧光显微镜下观察稳定表达 UBE2C的 AGS 胃癌细胞株（100×）
Figure 3. Stable expressing UBE2C of AGS gastric cancer cell strain under fluorescene microscope（100 ×）
A：Contrl-shRNA；B：UBE2C-shRNA2。
图 4 qRT-PCR 检测胃癌AGS细胞株中稳定表达UBE2C的水平
与对照组比较，*P < 0.05，n = 3。
Figure 4. stable expression of UBE2C in AGS gastric cancer cell strain determined by qRT-PCR
图 5 RTCA 实时监测胃癌AGS细胞增殖结果
与对照组比较，*P < 0.05，**P < 0.01，***P < 0.001，n = 3。
Figure 5. RTCA monitoring the proliferation of gastric cancer AGS cells
图 6 CCK-8 检测UBE2C对胃癌AGS细胞增殖的影响与对照组比较，***P < 0.001，n = 3。
Figure 6. Effect of UBE2C on proliferation of gastric cancer AGS cells detected by CCK-8
图 7 RTCA 实时监测胃癌AGS细胞迁移结果（*P < 0.05，***P < 0.001，n = 3）
Figure 7. RTCA monitoring the migration of gastric cancer AGS cells
*P < 0.05，***P < 0.001，n = 3。
表 1 引物信息
Table 1. Primer information
基因 引物序列5′→3′ UBE2C forward：ATCCATGGAGCAGCTGGAAC reverse：GGCAGCATGTGTGTTCAAGG GAPDH forward：GAGCCACATCGCTCAGACAC reverse：CATGTAGTTGAGGTCAATGAAGG 
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