摘要
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。
Objective To screen specific small interfering RNA（siRNA） target human BMP9 gene and to prepare recombinant adenovirus vector AdsiBMP9 for investigation of 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 lipofectamine transfection reagent, screened the effective in- terfering 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 MTY assay. Results The recombinant plasmid Pses-Hus-siBMP9 and recombinant adenovirus AdsiBMP9 were success- fully constructed and its titer was 1 × 1010IU/mL. Compared to the negative and non-infected controls, the expres-sion of BMP9 gene was significantly inhibited after the SK-BR-3 cells were infected by AdsiBMP9. SK-BR-3 cells infected with AdsiBMP9 showed a promotive effect. On the fifth day, the growth rate of experiment groups was sig- nificantly higher than that of negative groups. Conclusions Specific siRNA targeting human BMP9 gene was suc- cessfully constructed, which can effectively inhibit endogenous expression of BMP9 in SK-BR-3cells and promote its proliferation.
出处
《基础医学与临床》
CSCD
北大核心
2013年第2期149-155,共7页
Basic and Clinical Medicine
基金
国家自然科学基金(81172017
30800658)
