Effects of RNAi Silencing GTPBP4 Gene on Proliferation and Apoptosis of Colon Cancer RKO Cells
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摘要:目的
探讨GTPBP4基因沉默后，RKO细胞增殖和凋亡等生物学行为的改变。
方法将慢病毒GTPBP4-siRNA及CON053阴性病毒转染结肠癌RKO细胞株，以Real-time PCR和Western blot检测敲减效率。Cellomics细胞计数检测细胞生长，MTT法检测细胞增殖，FACS法进行细胞周期检测，并进行细胞克隆形成实验，AnnexinⅤ-APC单染法流式细胞仪检测细胞凋亡。
结果慢病毒成功感染RKO细胞，mRNA和蛋白检测均显示GTPBP4基因敲减成功。GTPBP4基因敲减后，RKO细胞增殖速率受到显著抑制，MTT值比值（即增殖倍数）减小，G0/1、G2/M期细胞显著增多，S期明显减少，细胞克隆集落数目减少，凋亡峰值明显高于对照组，且峰值出现时间早于对照组。
结论GTPBP4基因可能通过促进肿瘤细胞增殖、抑制凋亡而影响结肠癌的发生发展。
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关键词:
- 结肠癌 /
- 慢病毒 /
- 人类GTP结合蛋白4 /
- 细胞增殖 /
- 细胞凋亡
Abstract:ObjectiveTo investigate the changes of RKO cell proliferation and apoptosis after GTPBP4 gene silencing.
MethodsGTPBP4-siRNA and negative control CON053 were successfully transfected into colon cancer RKO cell line. Real-time PCR and Western blot were used to detect the knock down efficiency. Cellomics cell count was used to detect cell growth, MTT method was used to detect cell proliferation, FACS method was used to detect cell cycle, and cell clone formation experiment was carried out, cell apoptosis was analysed by AnnexinⅤ-APC single staining flow cytometry.
ResultsSlow virus successfully infected RKO cells, mRNA and protein detection showed that the GTPBP4 gene was knocked down successfully. After GTPBP4 gene knocked down, the cell proliferation rate significantly inhibited, MTT value ratio (i.e., multiplication) decreased, G0/1 and G2/M phase cells increased, S phase decreased, cell colony number decreased, apoptosis peak value was significantly higher than that in the control group, and the peak value appears earlier than that of the control group.
ConclusionGTPBP4 gene may affect the occurrence and development of colon cancer by promoting tumor cell proliferation and inhibiting apoptosis.
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Key words:
- Colon cancer /
- Slow virus /
- GTPBP4 /
- Cell proliferation /
- Cell apoptosis
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图 1 荧光倒置显微镜观察siRNA慢病毒感染后(96h) RKO细胞的白光和荧光表达(×100)
Figure 1 The white light and fluorescent expression of RKO cells infected by siRNA lentiviral (96h) observed under fluorescence inverted microscope (×100)
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图 2 Western blot检测转染病毒后实验组与对照组RKO细胞中GTPBP4蛋白表达
Figure 2 GTPBP4 protein in control group and the experimental group detected by Western blot
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图 3 Cellomics细胞计数检测细胞生长结果图
Figure 3 Cell growth results detected by cellomics cell count
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图 5 Annexin V-APC单染法流式细胞仪检测细胞凋亡峰值图
Figure 5 The apoptotic peak figure of V-APC Annexin single staining flow cytometry detect
表 1 对照组与实验组各周期细胞数量统计表
Table 1 Cell number of each cycle in control group and experimental group and control group
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