Construction and screening effective sequence of shRNA targeting human trefoil factor 3 / 临床耳鼻咽喉头颈外科杂志

ABSTRACT

OBJECTIVE@#Trefoil factor 3 plays a pivot role in oncogenic transformation, growth and metastatic extension of solid tumours besides mucosal protection. We screened the best siRNA sequence targeting human TFF3 by the transient-transfection of the lentiviral mediated shRNA into thyroid carcinoma K1 cells which secrete TFF3 themselves.@*METHOD@#Four siRNA transcription template hairpin structure target potential sites in human TFF3 mRNA sequence(132,170,258 and 537 bp,seperately) were selected and synthesized,as well as one negative shRNA(shRNAC). After annealing in vitro, insert pLVX-shRNA-puro construct recombinant plasmid, then enzyme digestion and sequencing analysis. The lentiviral-shRNAs were transient-transfected into K1 cells. TFF3 mRNA and protein levels were test by real-time PCR and western blot respectively in K1 cells at 48h post transient-transfected.@*RESULT@#Genetic mutations in two sequences of shRNA1~2, so the follow-up study terminated. The TFF3 expression were obviously inhibited in K1 cells at 48 hours post transient-transfected of shRNA3 and shRNA4. TFF3 (258-276) showed the highest silencing efficiency (TFF3 mRNA reduced 60.67% and TFF3 protein reduced 56.44%, P < 0.01) when the transfection efficiency was 76.83%.@*CONCLUSION@#pLVX-shRNA-puro-TFF3 expression plamid were successfully constructed and the highest efficiency sequences were screened. All these laid a foundation for further study about the function of TFF3 gene.