shRNAs Aiming at Glycosyltransferase Inhibit Invasive and Proliferative Ability of LoVo Cell Line in vitro
- VernacularTitle:靶向糖基转移酶shRNA表达质粒对LoVo细胞侵袭和增殖能力的抑制作用
- Author:
Fuli HE
;
Qiang MA
;
Jian ZHANG
- Publication Type:Journal Article
- Keywords:
GnT-Ⅴ, RNAi, colorectal cancer, invasive ability
- From:
Progress in Biochemistry and Biophysics
2006;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
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 of GnT-Ⅴ were established by G418 screening. The mRNA and protein expression of GnT-Ⅴ were measured by semi- quantitative 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
