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目的构建丝裂原活化蛋白激酶(MEK)基因的小干扰RNA(siRNA)表达载体,观察其静默效应及对结肠癌细胞SW1116生长周期和DNA甲基化的影响。方法选择MEK不同靶点寡核苷酸片段.克隆到pGCsilencer真核表达载体中。脂质体转染SW1116细胞,荧光显微镜评估转染效率,G418筛选稳定表达siRNA的细胞。Western印迹法检测siRNA对MEK蛋白表达的静默效果。应用四甲基偶氮唑盐微量酶反应比色法检测细胞生长活力,流式细胞术分析细胞周期,甲基化特异性PCR和DNA测序检测p16~(INK4A)基因启动子甲基化状态。结果构建2个靶点的MEK重组质粒,分别转染和联合转染SW1116细胞,转染效率约为72.1%,G418筛选培养2周得到稳定表达MEK siRNA的细胞,MEK蛋白抑制率分别为74.2%和69.1%和90.2%.下游分子ERK蛋白磷酸化水平随之降低;细胞增殖活力下降2～4倍和细胞周期阻滞于G1期,细胞周期负调控因子p16~(INK4A)启动子区呈低甲基化状态。结论MEK特异性siRNA表达载体对结肠癌细胞MEK蛋白表达有一定的静默效果,多靶点联合效果更好.阻滞细胞周期于G1期,促进p16~(INK4A)基因去甲基化。
Objective To construct small interfering RNA (siRNA) expression vector of mitogen - activated protein kinase (MEK) gene and observe the effect of silencing effect on the growth cycle and DNA methylation of colon cancer cell line SW1116. Methods The oligonucleotide fragments of different targets of MEK were selected and cloned into pGCsilencer eukaryotic expression vector. The liposomes were transfected into SW1116 cells, the transfection efficiency was evaluated by fluorescence microscopy, and the cells stably expressing siRNA were screened by G418. Western blotting was used to detect the silencing effect of siRNA on MEK protein expression. The cell viability was detected by MTT colorimetric assay, cell cycle was analyzed by flow cytometry, and methylation status of p16 INK4A promoter was detected by methylation-specific PCR and DNA sequencing. Results Two MEK recombinant plasmids were constructed and transfected into SW1116 cells respectively. The transfection efficiency was about 72.1%. After MEK siRNA was stained with G418 for 2 weeks, the MEK protein inhibition rates were 74.2% And 69.1% and 90.2%, respectively. The phosphorylation of ERK protein in downstream molecules was decreased, the cell proliferation activity decreased by 2 ~ 4 folds and the cell cycle arrest in G1 phase. The promoter region of INK4A promoter was negative Methylation status. Conclusion MEK-specific siRNA expression vector has a certain silencing effect on the expression of MEK protein in colon cancer cells, and the multi-target combination is more effective, arresting the cell cycle in G1 phase and promoting the demethylation of p16 INK4A gene.