RNAi技术对APMCF1基因表达的抑制作用及其对HepG2细胞的影响

The inhibitory effect of RNAi on APMCF1 gene and its effects on HepG2 cells

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 inhibit the expression of APMCF1 gene with RNAi and explore its effects on cell growth and cell cycle of HepG2 cells. Methods： According to the gene sequence and secondary structure of APMCFI, We designed the siRNAs targeting APMCF1 gene and cloned into siRNAs expmssion vector pSUNeo. The constructed piasmids were stably trausfected into HepG2 cells by lipofectamiue. The expression of APMCF1 mRNA in HepG2 ceils were examined by semi - quantitative RT - PCR, and its effects on cell growth and cell cycle were observed by MTT and flow cytometer（FCM）. Results： Semi - quantitative RT - PCR indicated that APMCF1 had been successfully inhibited in HepG2 cells. The growth speed of APMCF1 inhibited cells increased markedly. Analysis of cell cycle by FCM showed that 42.93% of the inhibited cells were in G1 phase and 40.25% in S phase, while 55.46% of the control HepG2 cells were in G1 phase and 32.42% in S phase. Conclusion： The inhibitory APMCF1 gene can increase the growth and cell cycle of human hepatocellular carcinoma cells HepG2. It maybe a cell cycle regulative gene.