论文部分内容阅读
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 human nasopharyngeal carcinoma cells. METHODS: Let-7 mimics were chemically synthesized and transiently transfected into nasopharyngeal carcinoma cells. The expression of let-7 in nasopharyngeal carcinoma cells was detected by fluorescence quantitative PCR. The effects of let-7 mimics on the proliferation of nasopharyngeal carcinoma cells were analyzed by cell counting and MTT assay. Scratch assay demonstrated the effect of let-7 overexpression on the migration of nasopharyngeal carcinoma cells. Flow cytometry was used to detect the effect of let-7 on cell cycle. Fluorescence quantitative PCR and Western blotting were used to detect the differential expression of cell cycle-related regulatory factors. RESULTS: let-7 mimics were expressed in NPC cells and increased with increasing concentrations of let-7 at 50 nmol / L. Cell counting and MTT showed that let-7 significantly inhibited cell proliferation after let-7 overexpression (P <0.05). Scratch assay showed that let-7 significantly inhibited nasopharyngeal carcinoma cell migration (P <0.05). The results of cell cycle showed that the proportion of G0-G1 phase in CNE-2 cells increased from 68.23% ± 2.03% to 82.90% ± 1.98% (P <0.05) and the proportion of S phase decreased from 22.07% ± 1.14% after let-7 overexpression To 5.80% ± 0.87% (P <0.05). The proportion of G2-M phase increased from 8.86% ± 1.10% to 11.46% ± 1.60%, P> 0.05. After let-7 overexpression, the mRNA levels of c-myc and CDK6 were down-regulated by 61.89% and 58.21%, respectively (P <0.05). The expression of c-myc and CDK6 protein in let-7 cells was also down-regulated by Western blotting (P <0.05). CONCLUSION: Overexpression of let-7 gene can significantly inhibit the proliferation of nasopharyngeal carcinoma cells. One of the mechanisms may be that NPC cells arrest G0-G1 phase by affecting the expression of c-myc and CDK6 genes.