let-7对鼻咽癌细胞增殖及迁移的影响

Effect of let-7 microRNA on cell proliferation and migration in nasopharyngeal carcinoma cells

目的:探讨let-7过表达对人鼻咽癌细胞增殖和迁移的影响。方法:化学合成let-7模拟物,瞬时转染鼻咽癌细胞,用荧光定量PCR法检测转染后let-7在鼻咽癌细胞中的表达。细胞计数和四甲基偶氮唑蓝(MTT)法分析转染let-7模拟物后对鼻咽癌细胞增殖的影响。划痕实验验证过表达let-7对鼻咽癌细胞迁移能力的影响。流式细胞术检测过表达let-7对细胞周期的影响,荧光定量PCR技术及Western blotting检测细胞周期相关调控因子的差异性表达。结果:let-7模拟物可在鼻咽癌细胞中表达,且随着其浓度的增加而增加,在50 nmol/L表达水平最高。细胞计数和MTT结果显示过表达let-7后能显著抑制细胞增殖(P<0.05),划痕实验证明let-7可显著抑制鼻咽癌细胞迁移(P<0.05)。细胞周期结果显示过表达let-7后CNE-2细胞使G0-G1期比例由68.23%±2.03%升高到82.90%±1.98%(P<0.05),S期比例由22.07%±1.14%降低到5.80%±0.87%(P<0.05),G2-M期比例由8.86%±1.10%升高到11.46%±1.60%,P>0.05。过表达let-7后,c-myc、CDK6的mRNA水平分别下调61.89%和58.21%(P<0.05)。Western blotting过表达let-7后c-myc及CDK6蛋白表达水平亦下调(P<0.05)。结论:过表达let-7基因可显著抑制鼻咽癌细胞增殖,其机制之一可能是通过影响细胞周期调节因子c-myc及CDK6等基因的表达使鼻咽癌细胞阻滞G0-G1期。

Objective： To investigate the effect of let-7 overexpression on the proliferation and migration of nasopharyngeal carcinoma（ NPC） cells. Methods： let-7 mimics were chemically synthesized and instantly transfected into nasopharyngeal carcinoma CNE-2 cells. The expression of let-7 in the NPC cells was quantified by real-time RT-PCR. The effect of over-expressed let-7 mimics on cell proliferation was analyzed by cell counting and MTT assay respectively. Scratch test was performed to assess the effect of over-expressed let-7 on NPC cell migration. Cell cycle was evaluated by flow cytometry. The expression of mRNA and protein levels of cell cycle regulators were examined by real-time RT-PCR and Western blotting. Results： The expression of let-7 mimics was found in NPC cells and increased in a dose-dependent manner,reaching a highest level at 50 nmol / L. Cell counting and MTT assay showed that over-expression of let-7 mimics significantly inhibited cell growth（ P 〈0. 05）. The scratch test showed significant inhibition of NPC cell migration by let-7. Compared with the control group,the percentage of cells in G0-G1 phase was increased from 68.23% ±2.03% to 82.90% ±1.98%（ P〈0.05）,and that in S phase were decreased from 22.07% ±1.14% to 5.80% ± 0.87%（ P 0.05）; however,the percentage of cells in G2-M phases was not statistically different,although it increased from 8. 86% ± 1. 10% to 11. 46% ±1. 60%（ P〉0.05）. The mRNA and protein expression levels of c-myc and CDK6 were decreased significantly,as indicated by Western blotting. Conclusion： Over-expression of let-7 may inhibit the growth of nasopharyngeal carcinoma cells in vitro. This may be underlain by a G0-G1 phase cell cycle arrest,which might be mediated by down-regulation of c-myc and CDK6.