In vitro study and in vivo application of microRNA to silence expression of the transforming growth factor β1 gene delivered to tenocytes.

Abstract

Objective To investigate the effects of delivery of microRNA(miRNA) to silence expression of the transforming growth factor β1(TGF β1) genes and its influence on expression of the type Ⅰ, Ⅲ collagen and connective tissue growth factor (CTGF) genes in cultured tenocytes and injured tendons. Methods Four miRNAs TGF β1 DNA and one sham sequence vector control were designed and synthesized according to chicken TGF β1 mRNA sequence. They were used to produce 5 plasmid expression vectors (miRNA #1, #2, #3, #4 and negative control). Cultured chicken tenocytes were transfected with these vectors. Under a fluorescence microscope, the expression of EGFP was examined, to evaluate the transfection efficiency of the vectors. The expression of TGF β1, collagen Ⅰ, Ⅲ, and CTGF genes were measured using real-time PCR. At week 1 and 6 post surgery, expression of TGF β1, collagen Ⅰ, Ⅲ, and CTGF gene were measured using real-time PCR. Results EGFP expression was confirmed in tendon cell using fluorescence microscope, which showed that 20%-25% of the tenocytes were transfected by the miRNA vectors. Compared with the negative control, expression of the TGF β1 gene in the tenocytes treated with miRNA #1 and #2 was decreased by 68% and 43%, respectively. In the cells treated with miRNA #1, expression of the type Ⅲ collagen and CTGF genes was significantly decreased by 70% and 68%, respectively. At week 1 post surgery, expression of the TGF β1 gene in the injured digital flexor tendons decreased by 67%, but expression of the collagen Ⅰ, Ⅲ, and CTGF genes did not change. At week 6, expression of the TGF β1 and type Ⅲ collagen genes was significantly decreased by 56% and 58%, respectively. Conclusion The study indicates that delivery of miRNA to silence expression of the TGF β1 gene may be a promising in reducing tendon adhesion formations. Key words: Gene silencing; Gene expression regulation; Transforming growth factor beta