Influence of long non-coding RNA HOTAIR on proliferation and apoptosis of gastric cancer cells by targeted regulation of miR-206
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摘要: 目的 探讨长链非编码RNA HOTAIR对人胃腺癌细胞(AGS)增殖、凋亡的影响。 方法 实时荧光定量聚合酶链式反应(PCR)检测正常胃上皮细胞(GES-1)与胃癌AGS细胞中HOTAIR的表达水平,以及si-RNA HOTAIR(si-HOTAIR)转染胃癌AGS细胞后HOTAIR及微小RNA-206(miR-206)的表达水平。生物信息学预测HOTAIR与miR-206的结合位点,荧光素酶报告实验验证二者靶向关系。将细胞分为对照组、si-HOTAIR组、si-HOTAIR+miR-206抑制剂组,采用CCK-8、平板克隆形成实验检测各组细胞的活性和增殖能力,流式细胞术检测各组细胞的凋亡水平,免疫印迹实验检测相关抗凋亡蛋白CDK9的表达水平。 结果 与正常人胃黏膜上皮GES-1细胞相比, AGS细胞中HOTAIR的表达量显著增高(P<0.001), miR-206的表达水平显著降低(P<0.01)。转染si-HOTAIR的AGS细胞中HOTAIR表达量显著降低(P<0.01), miR-206表达水平显著增高(P<0.01)。实验结果显示, miR-206模拟物(miR-206 mimics)能显著降低野生型HOTAIR质粒的荧光素酶活性(P<0.01)。与对照组相比, si-HOTAIR组细胞增殖能力显著降低,凋亡能力显著提高(P<0.001); 与si-HOTAIR组相比, si-HOTAIR+miR-206抑制剂组细胞增殖能力显著增高,凋亡能力显著降低(P<0.01)。实验结果显示, si-HOTAIR能显著降低CDK9的蛋白水平表达, miR-206抑制剂能显著减弱si-HOTAIR对CDK9表达的抑制作用。 结论 HOTAIR能通过靶向调控miR-206促进胃癌细胞的增殖,抑制胃癌细胞的凋亡。Abstract: Objective To investigate the influence of long non-coding RNA HOTAIR on proliferation and apoptosis of human gastric adenocarcinoma cells(AGS). Methods The expression level of HOTAIR in normal gastric epithelial cells(GES-1)and gastric cancer AGS cells as well as the expression levels of HOTAIR and microRNA-206(miR-206)in gastric cancer AGS cells transfected with si-RNA HOTAIR(si-HOTAIR)were detected by real-time fluorescence quantitative polymerase chain reaction(PCR). Bioinformatics was used to predict the binding site of HOTAIR and miR-206, and the targeting relationship between HOTAIR and miR-206 was verified by luciferase reporter assay. The cells were divided into control group, si-HOTAIR group, and si-HOTAIR plus miR-206 inhibitor group. CCK-8 and colony formation assay were used to detect the cell viability and proliferation ability in each group, apoptosis of cells was detected by flow cytometry, and immunoblot assay was used to detect the expression of related anti-apoptotic protein CDK9. Results Compared with normal GES-1 cells, the expression of HOTAIR in AGS cells was significantly higher(P<0.001), while expression of - miR-206 was significantly lower(P<0.01). The expression of HOTAIR in AGS cells transfected with si-HOTAIR significantly reduced(P<0.01), and the expression of miR-206 significantly increased(P<0.01). Experimental results showed that miR-206 mimics can significantly reduce the luciferase activity of the wild-type HOTAIR plasmid(P<0.01). Compared with the control group, the cellular proliferation ability of si-HOTAIR group significantly reduced, while the apoptosis ability significantly improved(P<0.001). Compared with the si-HOTAIR group, the cellular proliferation ability of si-HOTAIR plus miR-206 inhibitor group significantly increased, while apoptosis ability significantly reduced(P<0.01). Experimental results showed that si-HOTAIR can significantly reduce the expression of CDK9 protein, and miR-206 inhibitor can significantly reduce the inhibition function of si-HOTAIR on CDK9 expression. Conclusion HOTAIR can promote the proliferation of gastric cancer cells and inhibit the apoptosis of gastric cancer cells by targeted regulation of miR-206.
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Keywords:
- long non-coding RNA /
- HOTAIR /
- microRNA-206 /
- gastric adenocarcinoma /
- proliferation /
- apoptosis /
- luciferase activity /
- bioinformatics
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