Huang X, Jiao S F, Zhu F C, Liu D, Yi B
Department of Pharmacology and Molecular Therapeutics, Nanchang University School of Pharmaceutical Science, Nanchang 330006, China.
2nd Department of Abdominal Surgery, Jiangxi Provincial Tumor Hospital, Nanchang 330029, China.
Zhonghua Zhong Liu Za Zhi. 2016 Feb;38(2):93-9. doi: 10.3760/cma.j.issn.0253-3766.2016.02.004.
To construct a RNA interference lentiviral vector aimed at human ARK5 (AMPK-related protein kinase 5) gene and explore its effect on the biologic behavior of human gastric cancer SGC7901 cells.
Targeting human ARK5 mRNA coding sequence, we designed three specific short hairpin RNAs (shRNAs) and constructed the lentiviral vector, then infected human gastric cancer SGC7901 cells with this vector. Afterwards, we used qPCR and Western blot for detecting the silencing effect on ARK5 gene, MTT colorimetric assay to measure the cell proliferation, cell scratch test for cell migration and Transwell for cell invasion, and flow cytometry analysis for apoptosis in cells treated with glucose starvation and TNF-α.
Sequencing proved that the recombinant lentiviral vector containing ARK5-shRNA-3 was constructed successfully. Real time fluorescent quantitative PCR assay showed that the expression abundance of ARK5 gene in the normal control group, negative control group and ARK5-shRNA-3 infected group were 1.002+ 0.082, 1.001+ 0.050 and 0.140+ 0.003, respectively, showing a statistically significant difference (P<0.01). Cell scratch test showed that the cell migration rate of ARK5-shRNA-3 infected group was (38.5+ 4.3)%, significantly lower than that of the normal control group [(72.4+ 6.4)%] and negative control group [(75.1+ 7.1)%, P<0.01]. The results of Transwell test showed that the number of penetrating cells in the normal control group, negative control group and ARK5-shRNA-3 transfection group were 257.4±12.3, 245.7±11.6, 112.5±7.8, with a significant difference (P<0.01). After glucose starvation and TNF-α-treatment for 24 h, the cell death rate of the normal control group, negative control group and ARK5-shRNA-3 group were (11.7±3.2)%, (12.3±2.6)% and (30.8±4.3)%, respectively, showing that the cell apoptosis rate of ARK5-shRNA-3 transfected group was significantly higher than that of the normal control and negative control groups (P<0.01).
We have successfully constructed a recombinant lentiviral vector which can efficiently silence ARK5 gene. Using it we can inhibit the proliferation, migration, invasion of tumor cells, and promote cell apoptosis under the condition of TNF-α treatment and glucose starvation.
构建针对人ARK5（AMPK相关蛋白激酶5）基因的RNA干扰慢病毒载体，并探讨其对人胃癌SGC7901细胞生物学行为的影响。
针对人ARK5 mRNA编码序列设计3条特异性短发夹RNA（shRNAs），构建慢病毒载体，并用该载体感染人胃癌SGC7901细胞。之后，采用qPCR和蛋白质免疫印迹法检测对ARK5基因的沉默效果，MTT比色法检测细胞增殖，细胞划痕试验检测细胞迁移，Transwell检测细胞侵袭，流式细胞术分析葡萄糖饥饿和TNF-α处理后细胞的凋亡情况。
测序证明成功构建了含ARK5-shRNA-3的重组慢病毒载体。实时荧光定量PCR检测显示，正常对照组、阴性对照组和ARK5-shRNA-3感染组ARK5基因表达丰度分别为1.002±0.082、1.001±0.050和0.140±0.003，差异有统计学意义（P<0.01）。细胞划痕试验显示，ARK5-shRNA-3感染组细胞迁移率为（38.5±4.3）%，明显低于正常对照组[（72.4±6.4）%]和阴性对照组[（75.1±7.1）%，P<0.01]。Transwell试验结果显示，正常对照组、阴性对照组和ARK5-shRNA-3转染组穿膜细胞数分别为257.4±12.3、245.7±11.6、112.5±7.8，差异有统计学意义（P<0.01）。葡萄糖饥饿和TNF-α处理24 h后，正常对照组、阴性对照组和ARK5-shRNA-3组细胞死亡率分别为（11.7±3.2）%、（12.3±2.6）%和（30.8±4.3）%，表明ARK5-shRNA-3转染组细胞凋亡率明显高于正常对照组和阴性对照组（P<0.01）。
成功构建了能有效沉默ARK5基因的重组慢病毒载体。利用该载体可抑制肿瘤细胞的增殖、迁移和侵袭，并在TNF-α处理和葡萄糖饥饿条件下促进细胞凋亡。
Zotero 插件