Effectiveness of microRNA in Down-regulation of TGF-beta gene expression in digital flexor tendons of chickens: in vitro and in vivo study.

PURPOSE

Transforming growth factor (TGF)-beta is considered to be responsible for the formation of scars such as adhesions around healing digital flexor tendons. We proposed to deliver microRNAs (miRNAs) to silence expression of the TGF-beta1 gene and to investigate the effectiveness of miRNAs in down-regulation of the TGF-beta1 gene in vitro and in vivo.

METHODS

We designed and engineered 4 miRNAs according to genetic sequences of chicken TGF-beta1. Four plasmid vectors harboring the respective engineered miRNAs and 1 control vector were constructed. We transfected 30 wells of cultured tenocytes with these vectors and harvested them 48 hours later. The gene expression levels were quantified using real-time polymerase chain reactions. Subsequently, the miRNA that most effectively silenced TGF-beta gene in vitro was tested on 25 chickens in vivo. The miRNA and control vectors were injected into the injured tendons, respectively. At 1 and 6 weeks after surgery, the tendons were analyzed for gene expression and protein production.

RESULTS

In both in vitro and in vivo settings, delivery of miRNA to the tendon substantially down-regulated expression of the TGF-beta gene but did not affect expression of the collagen I gene. In the healing tendon, TGF-beta gene expression was significantly down-regulated by 50% to 60% at 1 and 6 weeks. At 6 weeks, the collagen III gene expression was significantly down-regulated by 55% at 6 weeks and the connective tissue growth factor gene was significantly down-regulated by 25%. At 6 weeks, TGF-beta protein was substantially decreased.

CONCLUSIONS

MicroRNA significantly down-regulates expression of the TGF-beta in vitro and in vivo. Application of miRNA did not down-regulate expression of the collagen I, but downregulated the collagen III gene. Application of miRNA treatment to modulate TGF-beta expression holds great promise in preventing tendon adhesion formation.