OBJECTIVETo investigate fibronectin synthesis in SD rat mesangial cells after transforming growth factor-beta1 (TGF-beta1) is silenced by the short interfering RNA (siRNA) expressed by reconstructed pGEFP-C1 vectors.

METHODSDepending upon the 538th - 556th (A) and 895th - 913th (B) nucleotides of rat TGF-beta1 gene, a nucleotide (A or B) was constructed into a small hairpin nucleotide which was separately (A or B) or together (A plus B) inserted into a pGEFP-C1 vector with three reconstructed pGEFP-C1 vectors separately expressing the siRNAs for A or/and B. TGF-beta1 and fibronectin were dynamically investigated for their interrelationship by ELISA in the supernatant and RT-PCR in their extracted total RNA.

RESULTSThe siRNA hairpin-like molecules were constructed according to the 538th - 556th nucleotides of rat TGF-beta1 gene were able to markedly silence the expression of TGF-beta1 mRNA (P < 0.01) and protein (P < 0.01) at 48 h. Lipfectamin 2000 transfection stimulated the peak secretion of fibronectin at 24 h in the control and the experimental group whose TGF-beta1 was not silenced, but the silence of TGF-beta1 in both experimental groups delayed the top values of fibronectin to 48 h (P < 0.01).

CONCLUSIONThe silence of TGF-beta1 by siRNA decreased the fibronectin expression, but the latter was possibly not completely TGF-dependent.