靶向同源盒A10特异性干扰性小RNA序列的筛选及功能鉴定

Screening and verification of the siRNA targeted for the HOXA10 gene

目的筛选有效干扰同源盒（HOX）A10基因表达的特异性干扰性小RNA（siRNA）序列并鉴定其功能，为利用RNA干扰技术靶向HOXA10防治肿瘤提供实验依据。方法设计合成3对针对HOXA10基因不同位点的siRNA序列，应用阳离子脂质体介导转染人肺腺癌A549细胞，采用反转录-聚合酶链反应（RT—PCR）检测HOXA10 mRNA表达，以HOXA10与β-actin灰度比值（ODR）表示相对表达水平。筛选出抑制HOXA10效果最佳的siRNA序列，应用四甲基偶氮唑盐（MTT）和流式细胞术分别检测该siRNA干扰HOXA10后对A549细胞增殖和凋亡的影响。结果3对siRNA均能抑制HOXA10的表达，其中siRNA1抑制HOXA10 mRNA的表达最为明显，ODR为（20．190±1．698）％；siRNA1对A549细胞的增殖抑制率可达（69．793±2．092）％；siRNA转染后细胞凋亡率显著提高，siRNA1诱导A549凋亡作用最为明显，凋亡率为（29．593±2．670）％。结论筛选出的siRNA1序列可有效沉默HOXA10基因的表达，并能有效抑制A549细胞增殖、促进其凋亡。

Objective To obtain effective siRNA fragment of HOXA10 gene and verify its function,to supply experimental evidence for tumor prevention and curation by RNAi targeting to HOXA10 gene.Methods Three pairs of small interfering RNA targeting to the different sites of HOXA10 were designed and introduced into A549. The mRNA expression of HOXA10 of A549 was detected by semi-quantitative RT-PCR, the cell proliferation was assayed by MTT, the apoptosis was measured by flow cytometry. ＇ The most effective siRNA assay was screened and was tested the relationship between it and proliferation and apoptosis. Results The mRNA of HOXA10 was inhibited by three siRNAs in A549 ceils, among which siRNA1 gave the strongest inhibiting of HOXA10 by ODR was （20.190±1.698） %. The inhibitory rate of cell proliferation was （69.793±2.092） % and the apoptosis rate was （29.593±2.670） %. Conclusion siRNA1 can specifically degrade HOXA10 mRNA and inhibit the proliferation of A549 cell and promote its apoptosis.