| 周淑亭,王 丽,黄玉红.eEF1A1低表达对肝癌细胞凋亡、增殖与迁移的影响及eEF1A1/A2与ANXA7间的相互作用[J].肿瘤学杂志,2018,24(8):755-763. |
| eEF1A1低表达对肝癌细胞凋亡、增殖与迁移的影响及eEF1A1/A2与ANXA7间的相互作用 |
| Effects of Down-regulating eEF1A1 Gene on Apoptosis，Proliferation and Migration of Hepatocarcinoma Cells and Interaction between eEF1A1/A2 and ANXA7 |
| 投稿时间：2017-04-17 |
| DOI：10.11735/j.issn.1671-170X.2018.08.B001 |
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| 中文关键词: eEF1A1 eEF1A2 ANXA7 肝肿瘤 |
| 英文关键词:eEF1A1 eEF1A2 ANXA7 hepatocarcinoma |
| 基金项目: |
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| 中文摘要: |
| 摘 要：［目的］研究shRNA抑制eEF1A1表达对肝癌Hca-P细胞凋亡、增殖、迁移的影响，并通过分别下调eEF1A1和ANXA7的表达，探讨肝癌Hca-P细胞中eEF1A1、eEF1A2与ANXA7表达的相关性及潜在的相互作用。［方法］ 构建靶向eEF1A1基因的shRNA质粒表达载体，瞬时转染至小鼠肝癌Hca-P细胞中，设无处理组（CON）、eEF1A1-shRNA组(eEF1A1-shRNA)、转染pGPU/GFP/Neo-NC的阴性对照组（NC）三组细胞进行实验。采用实时荧光定量PCR和Western blot技术检测eEF1A1的抑制效果，并分别检测各组eEF1A2、ANXA7在mRNA和蛋白水平的表达。CCK-8法、流式细胞仪、Transwell小室分别检测对细胞增殖、凋亡、迁移的影响。此外，复苏已稳定低表达ANXA7的Hca-P细胞，通过实时荧光定量PCR和Western blot技术检测无处理组（CON)、ANXA7-shRNA转染组（ANXA7-shRNA）、阴性对照组（NC）三组Hca-P细胞中eEF1A1、eEF1A2在mRNA和蛋白水平的表达。［结果］ eEF1A1靶向shRNA成功下调小鼠肝癌Hca-P细胞中eEF1A1基因的表达。流式细胞仪检测显示，CON组、eEF1A1-shRNA组、NC组细胞凋亡率分别为2.85%±0.37%、4.71%±0.21%和3.01%±0.33%，eEF1A1-shRNA组的细胞凋亡较CON组和NC组明显增加(P<0.05)；CCK-8实验发现，eEF1A1-shRNA转染细胞后细胞增殖能力较CON组和NC组明显下降；Transwell实验显示，CON组、eEF1A1-shRNA组、NC组迁移细胞数目为123.75±9.26、66.84±17.47、115.31±12.59，eEF1A1-shRNA组细胞迁移能力较CON组和NC组显著减弱（P<0.05）。qRT-PCR和Western blot检测显示，eEF1A1-shRNA组eEF1A2、ANXA7在mRNA和蛋白水平的表达量较CON组和NC组明显降低(P<0.05)，相较其无处理组的蛋白表达水平，eEF1A2和ANXA7分别下降了57%和21%；ANXA7-shRNA组中eEF1A1、eEF1A2在mRNA和蛋白水平的表达量较CON组和NC组亦明显降低(P<0.05)。［结论］ 通过靶向eEF1A1的shRNA使eEF1A1低表达，可诱导细胞凋亡，抑制细胞的增殖能力及迁移能力。eEF1A1、eEF1A2与ANXA7的表达密切相关，且eEF1A1与ANXA7存在相互作用，可能共同参与调控肿瘤的发生与发展。 |
| 英文摘要: |
| Abstract：［Objective］ To investigate the effect of down-regulating eEF1A1 gene on the apoptosis，proliferation and migration of hepatocarcinoma cells(Hca-P)，and the interactions between eEF1A1/A2 and ANXA7. ［Methods］ The mouse Hca-P cells were transfected with shRNA plasmid vector targeting eEF1A1 gene(eEF1A1-shRNA group) or pGPU/GFP/Neo-NC(NC group)，and the untransfected cells were set as CON group. The mRNA and protein expression levels of eEF1A2 and ANXA7 were detected by qRT-PCR and Western blot，respectively. Cell proliferation，apoptosis，migration after transfection were evaluated by CCK-8 method，flow cytometry and Transwell chamber，respectively. The pre-retained Hca-P cells with stably low expressing ANXA7 were revived，and the mRNA and protein expression levels of eEF1A1 and eEF1A2 in Hca-P cells were detected with qRT-PCR and Western blot，respectively in 3 groups. ［Results］ The shRNA targeting eEF1A gene successfully knocked down the mRNA and protein expression levels of eEF1A1. Flow cytometry assay suggested that the apoptotic rates were 2.85%±0.37%(CON)，4.71%±0.21%(eEF1A1-shRNA) and 3.01%±0.33%(NC)(P<0.05). CCK-8 assay showed that compared with CON and NC groups，the cell proliferation significantly declined in eEF1A1-shRNA group(P<0.05). The Transwell assay showed that cell migration in eEF1A1-shRNA group was lower than that of CON and NC groups(66.84±17.47 vs 123.75±9.26 and 115.31±12.59；P<0.05). The qRT-PCR and Western blot results showed that compared with the CON group and NC group，the expression levels of eEF1A2 and ANXA7 in eEF1A1-shRNA group were significantly decreased(P<0.05). Compared to the expression in untreated group，the protein expression levels of eEF1A2 and ANXA7 decreased by 57% and 21% respectively. The expression of eEF1A1 and eEF1A2 at mRNA and protein levels in ANXA7-shRNA group were also significantly lower than those of CON group and NC group(P<0.05). ［Conclusion］ Knocking down of eEF1A1 expression can induce apoptosis，inhibit proliferation and migration in Hca-P cells. It is suggested that the expression of eEF1A1，eEF1A2 and ANXA7 are closely related；there is interaction between eEF1A1 and ANXA7，which may be jointly involved in the regulation of tumor occurrence and development. |
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