摘要
通过构建针对N-乙酰氨基葡萄糖转移酶Ⅴ(GnT-Ⅴ)的小片段发夹状RNA(shRNA)干扰表达质粒,研究了shRNA表达质粒沉默GnT-Ⅴ基因后对LoVo细胞增殖、黏附以及迁移、侵袭能力的影响.设计了靶向GnT-Ⅴ基因的小干扰RNA(siRNA)靶序列,构建shRNA表达载体并转染人结肠癌LoVo细胞,通过G418筛选建立稳定低表达GnT-Ⅴ基因的细胞株.分别采用半定量逆转录聚合酶链反应(RT-PCR)和蛋白质印迹(Western blot)检测shRNA对GnT-Ⅴ基因mRNA及蛋白质表达的影响.并通过CCK-8增殖实验、异质黏附实验、划痕愈合实验、趋化运动实验、细胞侵袭实验评价pGPU6/GFP/Neo GnT-ⅤshRNA对人结肠癌LoVo细胞增殖、黏附以及迁移、侵袭能力的影响.实验成功地构建了GnT-Ⅴ shRNA表达质粒,并且该质粒明显下调GnT-Ⅴ的表达,LoVo GnT-Ⅴ/1564和LoVo GnT-Ⅴ/2224的mRNA水平的抑制率分别为82%和71.5%,蛋白质水平的抑制率分别为68%和56%.选择干扰效率较高的LoVo GnT-Ⅴ/1564进行进一步实验.CCK-8增殖实验显示,与阴性对照组相比,LoVo GnT-Ⅴ/1564的增殖受到明显抑制(P<0.001),尤以72h为著;下调GnT-Ⅴ表达可增强LoVo细胞的黏附能力(t=-3.357,P<0.01),而显著抑制LoVo细胞的趋化运动能力(t=44.051,P<0.001);划痕实验结果也显示抑制GnT-Ⅴ表达延长LoVo细胞的愈合时间;用Matrigel胶介导的细胞侵袭实验结果显示,LoVo GnT-Ⅴ/1564和LoVo GnT-Ⅴ/NC的穿膜细胞数分别为(5.10±1.25)个和(39.55±2.16)个,GnT-Ⅴ/1564组较阴性对照组明显减少(t=61.626,P<0.001).结果表明,靶向GnT-Ⅴ的shRNA真核表达质粒可以显著降低GnT-Ⅴ的表达,从而抑制LoVo细胞的增殖、迁移和侵袭能力,因此,该GnT-Ⅴ的siRNA序列可能成为治疗结直肠癌的有效靶点.
To construct expression vectors of small hairpin RNA aimed at N-acetylglucosaminyltransferase V （GnT- V ） gene, and to investigate effects of GnT- V shRNA on proliferation, adhesion, migration and invasion of LoVo cell line. siRNAs were designed according to the coding sequence of GnT- V gene, shRNA expression vectors were constructed and transfected into LoVo cell line, cell lines which stably expressed low level ofGnT-V were established by G418 screening. The mRNA and protein expression of GnT- V were measured by semiquantitative reverse transcription polymerase chain reaction（RT-PCR） and Western blot analysis, respectively. The effects of pGPU6/GFP/Neo GnT- V shRNA vectors on proliferation, adhesion, migration and invasion of LoVo cell line were evaluated by CCK-8 assay, heterogenous adhesion, wound closure assay, chemotactic migration and cell invasive experiment, respectively. GnT- V shRNA expression plasmid was constructed successfully and pGPU6/GFP/Neo GnT- V shRNA down-regulated expression of GnT- V dramatically in LoVo cell. Expression of LoVo GnT-V/1564 and LoVo GnT-V/2224 dereased by 82%, 71.5% respectively at mRNA level, and 68%, 56% respectively at protein level. The more effective interfered cell line, LoVo GnT-V/1564, was chosen to do further experiment. CCK-8 assay showed proliferation of LoVo GnT-V/1564 was suppressed obviously, compared to proliferation of negative control group cell （P 〈 0.001）,especially in 72 hours; down-regulation of GnT- V expression can enhance adhesive ability（t=-3.357, P 〈 0.01） and inhibit chemotactic migration（t=44.051, P 〈 0.001） in LoVo cell line; quantitative analysis of the wound closure assay also indicated that down-regulation of GnT- V expression can significantly prolong wound heal hours of LoVo cell line; cell invasive experiment using Matrigel gel showed that penetrative cell numbers of LoVo GnT- V/1564 and LoVo GnT- V/NC were 5.10±1.25 and 39.55±2.16 respectively, penetrative cell numbers of LoVo GnT- V/1564 cell was reduced obvio
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2009年第8期1071-1078,共8页
Progress In Biochemistry and Biophysics
基金
广东省科技计划项目(0054)~~
