转录因子Twist的siRNA筛选、腺病毒构建及功能检测

目的 筛选特异性沉默人的Twist基因的siRNA序列,构建siTwist腺病毒并在MG63及143B骨肉瘤细胞中进行功能鉴定.方法 体外退火获得4组siTwist双链DNA序列,克隆至含有Twist基因的pSOS-Twist质粒中获得pSOS-siTwist质粒,脂质体转染HEK293细胞,GFP检测筛选有功能的siTwist片段,将筛选出的siTwist序列构建腺病毒,感染143B骨肉瘤细胞.通过RT-PCR、Western 印迹检测Twist的表达.siTwist与Twist腺病毒共感染MG63骨肉瘤细胞,细胞计数及细胞侵袭实验检测siTwist对Twist的抑制作用.结果 在HEK293细胞中,4组siTwist中有2组GFP的表达明显降低,且siTwist腺病毒能抑制143B骨肉瘤细胞中内源性的Twist表达,Twist腺病毒能促进MG63骨肉瘤细胞的增殖和转移,而两组siTwist与Twist共感染组MG63细胞的增殖及迁徙率均明显低于Twist组（P〈0.05）.结论 筛选出两对特异性沉默Twist基因的siRNA片段,并成功构建腺病毒,转染细胞后能有效抑制内源性和外源性的Twist表达,为研究Twist在骨肉瘤细胞增殖和转移中的作用及具体机制提供了有效的分子工具.

Twist siRNA Screening, Adenoviral Vector Construction and Its Functional Identification

Objective To screen gene-specific siRNA of human Twist, construct Ad-siTwist and identify its function in MG63 and 143B osteosarcomatous cells. Methods Four pairs of doublestranded DNA fragments for silencing Twist gene were vector with Twist gene to get pSOS-siTwist plasmid. The annealed in vitro and cloned into pSOS-Twist four pSOS-siTwists plasmids were then transfected in HEK293 cells using Lipofectamine respectively, siRNA silence efficiency was selected by GFP detection. Afterwards, 143B cells were infected with constructed adenovirus siTwists. Expression of Twist was detected with RT-PCR and western blot. MG63 cells were co-infected by Ad-siTwist and Ad-Twist. Inhibition of Twist was checked by cell growth curve and migration-invasion assay. Results GFP expression was significantly reduced by two pairs of siTwist in HEK293 cells. Meanwhile, endogenous expression of Twist was inhibited by adenovirus-mediated siTwist in 143B cells. Twist promoted MG63 proliferation and migration. Cell growth and migration rate in coinfection groups were lower than that in Ad-Twist infection group （ P 〈 0. 01 ）. Conclusion Two gene-specific siRNA of human Twist gene and constructed the adenovirus were successfully selected, with effective inhibitive effects on both endogenous and exogenous Twist expression, which can be used for its further mechanism study in proliferation and migration of osteosareoma.