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肿瘤代谢与营养电子杂志
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酰基甘油激酶敲低对结直肠肿瘤细胞HCT116 增殖迁 移的影响
目的探讨AGK 敲低对人结肠肿瘤细胞HCT?116增殖、迁移的影响。方法本实验通过构建AGK 基因的shRNA沉默 慢病毒质粒,利用第二代慢病毒包装系统制备、浓缩慢病毒液,并感染人结肠肿瘤细胞HCT?116细胞株。经过嘌呤霉素抗性筛选 获得敲低AGK 基因的HCT?116细胞系。利用qRT?PCR 和Western blotting 检测AGK 敲低后在mRNA水平和蛋白水平表达情况; 利用CCK?8 试剂盒检测AGK 敲低稳定细胞株细胞增殖情况;利用划痕实验检测AGK 敲低后对细胞迁移的影响。结果本实验 构建的AGK 基因的shRNA 沉默慢病毒质粒可以有效敲低HCT116中内源性AGK。同野生型细胞株相比,AGK 敲低稳转株 HCT116?ASR?876和HCT116?ASR?878细胞中AGK 的mRNA 水平和蛋白水平表达显著地降低(P <0.001)。通过CCK?8 检测发现, AGK 敲低后显著降低细胞增殖能力(P<0.05)。划痕实验表明,AGK 敲低能显著抑制细胞迁移作用。结论shRNA慢病毒感染后, HCT?116细胞中AGK 基因的表达量显著低于野生型细胞株,并且初步验证了AGK 可以促进HCT116 细胞的增殖和迁移。
The effects of knockdown AGK gene on proliferation and migration capacity of colorectal cancer cells
Objective In order to investigate the effects of acylglycerol kinase(AGK) on proliferation and migration in HCT116 cells. Methods In this study, shRNA silencing lentivirus plasmid of AGK gene was constructed, and the second generation lentivirus packaging system was used to prepare and concentrate lentivirus venom and infect human colon cancer cell HCT?116 cell line.Then HCT?116 cell lines with knockdown AGK gene were screened for puromycin resistance.qRT?PCR and Western blotting were used to detect the mRNA and protein expression of AGK after knockdown. CCK?8 kit was used to detect the proliferation of AGK knockdown stable cell lines.The effect of AGK knockout on cell migration was detected by cells scratch assay. Results ShRNA silencing lentivirus plasmid of AGK gene constructed in this experiment can effectively knockdown endogenous AGK in HCT116.The mRNA and protein levels of AGK in HCT116?ASR?876 and HCT116?ASR?878 cells were significantly lower than those of wild?type cell lines (P<0.001).CCK?8 detection showed that AGK knockdown significantly reduced cell proliferation ability (P<0.05).The scratch test showed that AGK knockdown could significantly inhibit cell migration. Conclusion Knockout of AGK gene can significantly reduce the proliferation and migration abilities of HCT116 cells, which might provide a new potential target for targeted therapy of colorectal cancer.