人腺苷酸活化蛋白激酶5基因RNA干扰慢病毒载体的构建及其对人胃癌SGC7901细胞生物学行为的影响

Construction of a RNAi lentiviral vector targeting ARK5 gene and its effect on the biological behavior of gastric cancer SGC7901 cells

目的探讨人腺苷酸活化蛋白激酶5（ARK5）基因RNA干扰（RNAi）慢病毒载体的构建及其对人胃癌SGC7901细胞生物学行为的影响。方法以人ARK5mRNA编码序列为干扰靶点，设计3条沉默ARK5的特异性短发卡RNA（shRNA），构建重组慢病毒表达载体，转染人胃癌SGC7901细胞。采用实时荧光定量PCR和Westernblot检测ARK5基因的沉默效果，四甲基偶氮唑蓝（MTT）法检测细胞增殖能力，细胞划痕实验检测细胞迁移能力，Transwell法检测细胞侵袭能力，流式细胞仪检测葡萄糖饥饿和肿瘤坏死因子α（TNF—α）诱导处理对细胞凋亡的影响。结果测序结果证明，RNAi重组慢病毒载体ARK5-shRNA-3构建成功。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转染组的ARK5基因表达水平分别为1．002±0．082、1．001±0．050和0．140±0．003。ARK5．shRNA-3转染组与正常对照组、阴性对照组比较，差异有统计学意义（P〈0．01）。细胞划痕实验显示，ARK5-shRNA-3转染组的细胞迁移率为（38．5±4．3）％，而正常对照组和阴性对照组的细胞迁移率分别为（72．4±6．4）％和（75．1±7．1）％，差异有统计学意义（P〈0．01）。Transwell检测结果显示，正常对照组、阴性对照组和ARK5-shRNA-3转染组的穿膜细胞数分别为（257．4±12．3）个、（245．7±11．6）个、（112．5±7.8）个，ARK5-shRNA-3转染组与正常对照组、阴性对照组差异有统计学意义（P〈0．01）。在葡萄糖饥饿和TNF-α处理24h后，正常对照组、阴性对照组和ARK5-shRNA-3转染组的细胞凋亡率分别为（11．7±3．2）％、（12．3±2．6）％和（30．8±4．3）％，ARK5-shRNA-3转染组与正常对照组、阴性对照组差异有统计学意义（P〈0．01）。结论成功构建了能高效沉默ARK5基因的重组慢病毒表达载体，并且明显地抑制了胃癌细胞增殖、迁移和侵袭，促进了胃癌细胞在饥饿胁迫及TNF—α诱导情况下的凋亡。

Objective 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. Methods 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. Results 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 saormal 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）. Conclusions 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.