Objective   To construct a recombinant vector encoding RNA interference (RNAi) targeting focal adhesion kinase (FAK)

and explore the effect of FAK silencing on the formation of colon carcinoma multicellular spheroids (MCSs).    Methods   One specific pair of oligonucleotides containing short hairpin structure and their negative control sequence based on FAK cDNA sequences were designed and synthesized. After annealed

they were inserted into pGenesil-l vector to generate the recombinant plasmids. The recombinant plasmids were digested with

Eco

RⅠ and

Hin

dⅢ to execute the

restriction endonuclease identification

and then DNA sequencing was performed for sequence analysis. The recombinant plasmids were transfected into cultured colon carcinoma HT29 cells using lipofectamine

TM

2000. The stably transfected cells were selected in a medium containing geneticin G418. The change of FAK expression in HT29 cells before and after RNAi was detected by real-time polymerase chain reaction (real-time PCR) and Western blot analysis. The cells were cultured as suspended to observe the effect of RNAi targeting FAK on MCSs formation.    Results   The recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis. After RNAi targeting FAK

  FAK mRNA  and  protein expression were decreased significantly to (79.20±2.97)% and (78.47±4.39)% respectively

and the cells were not so easy to aggregate and form MCSs.    Conclusion   The recombinant vector of RNAi targeting FAK is constructed successfully

the expression of targeting gene FAK is inhibited markedly

and the formation of MCSs is inhibited efficiently.