Apollon特异性siRNA序列的筛选及功能鉴定

Screening and verification of the siRNA targeting Apollon

目的 筛选干扰Apollon基因表达的siRNA序列并鉴定其功能.方法 采用化学方法合成针对Apollon基因不同位点的siRNA序列;应用Lipofectamine 2000转染人类乳腺癌MCF-7细胞;反转录PCR（RT-PCR）检测Apollon mRNA表达水平;细胞免疫荧光结合激光共聚焦技术定量分析Apollon蛋白的表达;四甲基偶氮唑蓝（MTT）和流式细胞术分别检测siRNA干扰Apollon后对MCF-7细胞增殖和凋亡的影响.结果 合成的3对siRNA序列均能抑制Apollon mRNA表达,其siRNA1、siRNA2、siRNA3对ApollonmRNA的抑制率分别为（36.201±11.629）％、（67.308±7.686）％、（47.123±12.000）％,与对照组相比差异均有统计学意义（均P＜ 0.05）;siRNA2转染MCF-7细胞后Apollon蛋白的荧光强度为（14.97±2.08）％,增殖抑制率达（73.361±2.118）％,凋亡率为（28.793±0.743）％.结论 筛选出的siRNA2序列可有效沉默Apollon基因的表达,显著抑制MCF-7细胞的增殖和促进其凋亡,为肿瘤靶向治疗提供实验依据.

Objective To screen siRNAs that can effectively inhibit Apollon gene expression and determine the cellular functions of those siRNAs.Methods A chemical synthesis method was used to synthesize 3 siRNA sequences against different sites of Apollon.They were transfected into the human breast cancer MCF-7 cells by using Lipofectamine 2000.mRNA level of Apollon was determined by reverse transcription-polymerase chain reaction （RT-PCR）.Cellular immunity fluorescence quantitative analysis combined with confocal laser technology was used to determine the protein level of Apollon.Methyl thiazolyl tetrazolium bromide （MTT） assay and flow cytometry were used to determine the effects of siRNA targeting Apollon on proliferation and apoptosis of MCF-7 cells,respectively.Results Three pairs of siRNA could significantly inhibit Apollon mRNA expression,at the inhibition rates of （36.201±11.629） %,（67.308±7.686） %and （47.123±12.000） %,respectively （P 〈 0.05）.After tranfection by siRNA2,Apollon protein fluorescence intensity was （14.97±2.08） % compared with control cells.The cell proliferation MCF-7 was inhibited by （73.361±2.118） %and apoptosis was increased by （28.793±0.743） %.Conclusions Screened siRNA2 effectively silences Apollon gene expression,effectively inhibits the proliferation and increases the apoptosis of MCF-7 cells.This provids the foundation for its clinical application in cancer therapy.