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[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(qRT-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)%, P<0.05],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P<0.05]。转染miR-NA-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+pcDNA-CAR-MA3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,P<0.05)。miR-24+pcDNA3-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)%,P<0.05];而miR-24+pcDNA3-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)%,P<0.05;晚期凋亡率:(6.38±0.63)% vs (15.56±2.27)%,P<0.05]。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。
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[Abstract]
Objective:To explore effect of miRNA-24 on proliferation and apoptosis of the gastric cancer AGS cell and its potential mechanism. Methods: Expressions of miRNA-24 in gastric cancer AGS, MKN and HGC27 cells as well as gastric mucosal epithelium GES-1 cell were detected by qRT-PCR. The AGS cell line with over-expression of miRNA-24 was constructed. CCK-8, flow cytometry and Western blotting assays were respectively used to mea-sure proliferation vibility of the cells, cell cycle and apoptosis, and expressions of cell cycle and apoptosis-related cyclin D1, CDK2, Bcl-2, p-IκB-α/IκB-α, p-Rb/Rb proteins. Possible target gene of miRNA-24 was forecasted and verified with dual luciferase report gene assay. Results: Expressions of miRNA-24 in the gastric cells was obvious-ly lower than that in the GES-1 cell (P<0.05). Proliferation vibility of the AGS cell at 48 h in the transfected with miRNA-24 mimic (miR-24 ) group was remarkably lower than that in the control miR-NC group ([119.62 ± 12.63]% vs [147.79 ± 11.89]%, P<0.05), an arrest of cell cycle occurred, and its early and later apoptosis rates were evidently more increased than those in the miR-NC group(early apoptosis rate:[11.32+2.27]% vs [0.57 ± 0.08]%; later apopto-sis rate:([15.56 ± 2.27]% vs [0.85 ± 0.16]%, all P<0.05). In the miRNA-24 group, expression levels of cyclin D1,CDK2, Bcl2 and p-Rb proteins were all obviously lower than those in the miR-NC group, and expression level of p-IκB-α protein was obviously higher than that in the miR-NC group. In co-transfection of miRNA-24 mimic and over-expression plasmid of CARMA3 that may be action target of miRNA-24 (miR-24+pcDNA-CARMA3) group,CARMA3 protein expression of the AGS cell was significantly higher than that in the miR-24 group (1.74 ± 0.09 vs 1.03 ± 0.06, P<0.05). Proliferation vibility of the AGS cell at 48 h in the miR-24+pcDNA-CARMA3 group was obvi-ously higher than that in the miR-24 group ([137.85 ± 15.34]% vs [102.31 ± 11.23]%, P<0.05), however, early and lat-er apoptosis rates of the AGS cell in the miR-24+pcDNA-CARMA3 group were all lower than those in the miR-24 group (early apoptosis rate: [4.24 ± 0.56]% vs [11.32 ± 2.27]%, later apoptosis rate: [6.38+0.63]% vs [15.56 ± 2.27]%,all P<0.05). Effect of miRNA-24 on proliferation of apoptosis of the gastric cancer AGS cell can be partially re-versed by over-expression of CARMA3. Conclusion: miRNA-24 could inhibit proliferation of the gastric caner cells and promote their apoptosis through targeting CARMA3.
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