miRNA-24通过靶向CARMA3基因调控胃癌AGS细胞的增殖和凋亡

发布时间：2018-04-19 00:41

本文选题：微小RNA- + 胃癌细胞株AGS　；参考：《中国肿瘤生物治疗杂志》2017年10期

【摘要】：目的:探讨miRNA-24对胃癌AGS细胞增殖和凋亡的影响及其潜在的作用机制。方法:实时定量聚合酶链式反应(q RT-PCR)检测不同胃癌细胞株(AGS、MKN74、HGC27)和胃黏膜上皮GES-1细胞中miRNA-24的表达水平。建立miRNA-24过表达的AGS细胞株,采用CCK-8法检测细胞增殖活力,流式细胞术检测细胞周期和凋亡情况,Western blotting检测细胞周期和凋亡相关cyclin D1、CDK2、Bcl-2、p-IκB-α/IκB-α和p-Rb/Rb蛋白的表达水平;双荧光素酶报告基因分析法预测和验证miRNA-24可能的靶基因。结果:3株胃癌细胞中miRNA-24的表达均低于GES-1 cell。转染miRNA-24 mimic(miR-24组)48 h后AGS细胞增殖活力显著低于miR-NC组[(119.62±12.63)%vs(147.79±11.89)%,P0.05],并出现周期阻滞,且早期和晚期细胞凋亡率明显较miR-NC组上升[早期凋亡率:(11.32±2.27)%vs(0.57±0.08)%;晚期凋亡率:(15.56±2.27)%vs(0.85±0.16)%,均P0.05]。转染miRNA-24 mimic后,细胞中cyclin D1、CDK2、Bcl-2及p-Rb的蛋白表达水平均较miR-NC组显著降低,p-IκB-α蛋白的表达水平较miR-NC组显著上升。共转染miRNA-24 mimic和miRNA-24可能作用靶点CARMA3基因过表达质粒的miR-24+pc DNA-CARMA3组AGS细胞CARMA3蛋白表达较miR-24组明显增加(1.74±0.09 vs 1.03±0.06,P0.05)。miR-24+pc DNA3-CARMA3组AGS细胞48 h增殖活力较miR-24组显著升高[(137.85±15.34)%vs(102.31±11.23)%,P0.05];而miR-24+pc DNA3-CARMA3组AGS细胞早期凋亡率和晚期凋亡率均较miR-24组显著降低[早期凋亡率:(4.24±0.56)%vs(11.32±2.27)%,P0.05;晚期凋亡率:(6.38±0.63)%vs(15.56±2.27)%,P0.05]。CARMA3过表达可部分逆转miRNA-24对胃癌AGS细胞增殖及凋亡的作用。结论:miRNA-24可通过靶向CARMA3基因抑制胃癌细胞的增殖、促进其凋亡。
[Abstract]:Aim: to investigate the effect of miRNA-24 on proliferation and apoptosis of gastric cancer AGS cells and its possible mechanism.Methods: Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the expression of miRNA-24 in different gastric cancer cell lines (AGSN MKN74, HGC27) and gastric epithelial GES-1 cells.The miRNA-24 overexpression AGS cell line was established. The proliferative activity was detected by CCK-8 assay, and the cell cycle and apoptosis were detected by flow cytometry. The expression levels of cyclin D1- CDK2pBcl-2mp-I 魏 B- 伪 / I 魏 B- 伪 and p-Rb/Rb protein were detected by flow cytometry (FCM).Double luciferase reporter gene analysis was used to predict and verify the possible target genes of miRNA-24.Results the expression of miRNA-24 in the 3 strains of gastric cancer was lower than that in GES-1 cells.The proliferative activity of AGS cells in miRNA-24 mimic(miR-24 group was significantly lower than that in miR-NC group 48 h after transfection [119.62 卤12.63)%vs(147.79 卤11.89 + P 0.05], and cell cycle arrest was found, and the early and late apoptosis rate was significantly higher than that in miR-NC group [early apoptosis rate: 11.32 卤2.27)%vs(0.57 卤0.08; late apoptosis rate: 15.56 卤2.27)%vs(0.85 卤0.16, P0.05].After transfection of miRNA-24 mimic, the expression levels of Bcl-2 and p-Rb in cyclin D1- CDK2 and p-Rb were significantly lower than those in miR-NC group. The expression level of p-I 魏 B- 伪 protein was significantly increased compared with miR-NC group.The expression of CARMA3 protein in AGS cells of miR-24 PC DNA-CARMA3 group was significantly higher than that of miR-24 group in miR-24 PC DNA-CARMA3 group (1.74 卤0.09 vs 1.03 卤0.06) P0.05N. MiR-24 PC DNA3-CARMA3 group was significantly higher than miR-24 group [137.85 卤15.34)%vs(102.31 卤11.23], and miR-24 PC DNA3-CARMA3 group was AGS significantly higher than miR-24 group.The early apoptosis rate and late apoptosis rate were significantly lower than those in miR-24 group [the early apoptosis rate was 4.24 卤0.56)%vs(11.32 卤2.27; the late apoptotic rate was 6.38 卤0.63)%vs(15.56 卤2.27P 0.05] .CARMA3 overexpression could partially reverse the effect of miRNA-24 on the proliferation and apoptosis of gastric cancer AGS cells.Conclusion: miRNA-24 can inhibit the proliferation and promote apoptosis of gastric cancer cells by targeting CARMA3 gene.
【作者单位】：新乡医学院第一附属医院消化内科;
【分类号】：R735.2

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