siRNA干扰人BMP9重组腺病毒载体的构建及其促进乳腺癌SK-BR-3细胞增殖

 目的 筛选特异性干扰人BMP9基因的siRNA序列并制备重组腺病毒AdsiBMP9，探讨RNAi人BMP9基因后对乳腺癌SK-BR-3细胞增殖能力的影响。方法 设计制备3对干扰人BMP9的双链DNA序列，亚克隆至Pses-Hus质粒中获得Pses-Hus-siBMP9质粒，脂质体转染乳腺上皮细胞HBL-100筛选有效干扰质粒，构建重组腺病毒并感染SK-BR-3细胞，RT-PCR，Western blot 检测BMP9表达，MTT检测细胞增殖能力。 结果 成功构建并筛选出针对人BMP9基因的有效干扰质粒并包装成腺病毒，病毒滴度为1×1010IU/mL，感染SK-BR-3细胞显示BMP9在转录水平和翻译水平表达量显著低于对照组和空白组(P<0.05)，第5天AdsiBMP9组细胞的增殖率显著高于AdsiNC组（P<0.05）。结论 成功构建特异性沉默人BMP9基因的siRNA腺病毒载体，可有效抑制SK-BR-3细胞中BMP9基因的表达从而促进该细胞增殖。

Construction of recombinant adenovirus vector interfering the expression of human BMP9 and promoting the proliferation of breast cancer SK-BR-3 cells

Objective To screen specific small interfering RNA(siRNA) target human BMP9 gene and prepare recombinant adenovirus vector AdsiBMP9 then investigate its effects on the proliferation of breast cancer SK-BR-3cells. Methods Three pairs of double-stranded DNA fragments for silencing human BMP9 were designed and synthesized, then subcloned into the shuttle plasmid Pses-Hus. The recombinant plasmids Pses-Hus-siBMP9 were transfected into the breast epithelial cells HBL-100 by using lipofectamine transfection reagent, Screened the effective interfering plasmid, constructed AdsiBMP9 and infected SK-BR-3 cells. The expression level of BMP9 mRNA and protein were detected by RT-PCR and western blot. The proliferation of SK-BR-3 cells were observed with MTT assay. Results The recombinant plasmid Pses-Hus-siBMP9 and recombinant adenovirus AdsiBMP9 were successfully constructed and its titer was 1×1010IU/ml. Compared to the negative and non-infected controls, the expression of BMP9 gene was significantly inhibited after the SK-BR-3 cells were infected by AdsiBMP9(P<0.05). SK-BR-3 cells infected with AdsiBMP9 showed a promoted proliferation effect. On the fifth day, the growth rate of experiment groups was significantly higher than that of negative groups(P<0.05). Conclusion Specific siRNA targeting human BMP9 gene was successfully constructed, which can effectively inhibit endogenous expression of BMP9 in SK-BR-3cells and promote its proliferation.