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目的:采用RNAi抑制APMCF1基因表达,并观察其对HepG2细胞生长和细胞周期的影响。方法:根据APMCF1基因序列及其二级结构特征,设计并合成针对APMCF1基因的siRNA寡核苷酸,经退火形成双链后克隆入pSUNeo载体,并采用脂质体介导法将其稳定转染至人肝癌细胞系HepG2,RT-PCR检测siRNA对靶基因mRNA的抑制效果,然后采用MTT实验、流式细胞仪等对细胞的生长及细胞周期进行观察。结果:HepG2细胞的APMCF1基因表达被成功的抑制。APMCF1基因表达受抑制的HepG2细胞的生长速度加快;细胞周期发生改变,G1期细胞比例减少,S期细胞比例增多,差异具有显著性。结论:HepG2细胞的APMCF1基因表达被成功的抑制。APMCF1基因对HepG2细胞的生长和细胞周期有明显的调节作用,可能是一个侯选的细胞周期调节基因。
Objective: To suppress the expression of APMFC1 gene by RNAi and observe its effect on the growth and cell cycle of HepG2 cells. METHODS: According to the sequence of APMCF1 gene and its secondary structure characteristics, siRNA oligonucleotide targeting APMCF1 gene was designed and synthesized. After annealing, it was double-stranded and cloned into pSUNeo vector, and it was stably transfected by liposome-mediated method. To human hepatocellular carcinoma cell line HepG2, RT-PCR was used to examine the inhibitory effect of siRNA on target gene mRNA. Then MTT assay and flow cytometry were used to observe cell growth and cell cycle. RESULTS: The expression of APMCF1 in HepG2 cells was successfully inhibited. The growth rate of HepG2 cells with inhibited APMCF1 gene expression was accelerated; the cell cycle changed, the proportion of cells in G1 phase decreased, and the proportion of cells in S phase increased, the difference was significant. Conclusion: The expression of APMCF1 gene in HepG2 cells was successfully inhibited. APMCF1 gene has obvious regulation on the growth and cell cycle of HepG2 cells, and may be a candidate cell cycle regulator gene.