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miRNA-33a抑制人结肠癌细胞HCT-116增殖的机制研究
作者:黄重发 付启梅 周怿 朱清 卜全慧 
单位:210002  南京 解放军第454医院消化科 
关键词:微RNAs 结肠肿瘤 细胞增殖 
分类号:R574.6
出版年,卷(期):页码:2016,41(9):725-729
摘要:
[摘要] 目的 探讨miRNA-33a抑制人结肠癌细胞HCT-116增殖的机制。方法 化学法合成miRNA-33a mimicsmiRNA-33a inhibitor及阴性对照(NC)序列,然后转染至HCT-116细胞。转染后48h,采用Real-time PCR检测细胞中miRNA-33a含量的变化,Western blotting检测细胞中Twist蛋白含量的变化;转染后244872h,采用CCK-8的检测肿瘤细胞增殖活性的变化。采用信息学软件预测miRNA-33aTwist的结合位点,并通过荧光素酶报告基因法证实预测的结合位点。结果 miRNA-33a mimicsmiRNA-33a inhibitor转染后可明显上调或下调HCT-116细胞内miRNA-33a的相对含量(P<0.05),而NC转染组细胞miRNA-33a含量与对照组比较差异无统计学意义(P>0.05)miRNA-33a mimicsmiRNA-33a inhibitor转染后可明显下调或上调HCT-116细胞内Twist蛋白的表达,与对照组比较差异有统计学意义(P<0.05),而NC转染组细胞内Twist含量与对照组比较差异无统计学意义(P>0.05)miRNA-33a mimicsmiRNA-33a inhibitor转染后可明显下调或上调HCT-116细胞对数生长期的增殖活性,转染后48h72h与对照组比较差异均有统计学意义(P<0.05),而NC转染组细胞增殖活性与对照组比较差异无统计学意义(P>0.05)。荧光素酶实验显示,miRNA-33a mimicsmiRNA-33a inhibitor转染可以抑制或促进野生型报告基因荧光素酶活性(P<0.05),而对突变型报告基因荧光素酶活性无明显影响(P>0.05)结论 miRNA-33a可以通过抑制其靶基因Twist的表达抑制HCT-116细胞的增殖活性。

[Abstract]  Objective  To explore the mechanism of chemically synthesized miRNA-33a inhibiting proliferation activity of colon cancer cells HCT-116. Methods  HCT-116 cells were transfected with miRNA-33a mimics, miRNA-33a inhibitor and miRNA-33a negative control (NC). The contents of miRNA-33a in the transfected cells and changes of Twist protein expression were detected by real-time PCR and Western blotting respectively 48 hours after transfection; the changes in cell proliferation activity were measured using CCK-8 method 72 hours after transfection. Bioinformatic software was used to predict the binding site of miRNA-33a with Twist, and a luciferase reporter method was used to verify whether there is the target site of miRNA-33a in Twist gene. Results  Transfection of HCT-116 cells with miRNA-33a mimics significantly increased miRNA-33a mRNA and decreased Twist protein expression (P<0.05), but the transfection with miRNA-33a inhibitor significantly decreased miRNA-33a mRNA and increased Twist protein expression (P<0.05); moreover, miRNA-33a mimics significantly inhibited proliferation activity of HCT116 cells and miRNA-33a inhibitor significantly enhanced cell proliferation activity 48 and 72h after transfection compared with the control group (P<0.05). The binding site of miRNA-33a in targeting gene Twist was successfully predicted and verified. Conclusion  miRNA-33a inhibits the proliferation activity in HCT-116 cells by silencing the expression of its target gene Twist.

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