Relationship between the expression of focal adhesion kinase and the sensitivity of HT29 cells to 5-fluorouracil
- VernacularTitle:HT29细胞黏着斑激酶表达与其对5-氟尿嘧啶敏感性的关系研究
- Author:
Yuying CHEN
;
Li YANG
;
Feng PAN
- Publication Type:Journal Article
- Keywords:
focal adhesion kinase;
RNA interference;
colonic neoplasms;
gene expression
- From:
Medical Journal of Chinese People's Liberation Army
1981;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective RNA interference (RNAi) expression vector was constructed to inhibit the focal adhesion kinase(FAK)expression in colon carcinoma HT29 cells, and then the sensitivity of the cells to 5-fluorouracil (5-FU) was determined. Methods One specific pair of oligonucleotides with short hairpin and its negative control sequence were designed and synthesized based on FAK cDNA sequences, then they were inserted into pGenesil-l vector to generate the recombinant plasmids. After identification by the restriction endonuclease and DNA sequencing, the recombinant plasmids were transfected into HT29 cells by lipofectamine TM 2000. The stable transfected cells were selected in a medium containing geneticin G418. The change in FAK expression in HT29 cells before and after RNA interference was detected by reverse transcription and polymerase chain reaction (RT-PCR) analysis and SP immunocytochemistry technique. Sensitivity of HT29 cells to 5-FU was determined by MTT assay. Results The recombinant plasmids coincided completely with the designs in the restriction map and the sequence analysis. After FAK being targeted by RNA interference, immunocytochemistry showed the protein expression of FAK was reduced dramatically, and RT-PCR revealed FAK mRNA expression was down-regulated by 76.94% compared to that of untransfected cells. MTT assay also showed that the sensitivity of HT29 cells to 5-FU in transfected pGenesil-1-FAK vector cells was increased, while IC50 declined remarkably (P
