Design,synthesis and activity evaluation of small interfering RNA targeting connective tissue growth factor

Objective To design, synthesize and screen high efficient small interfering RNA(siRNA) targeting to connective tissue growth factor (CTGF) in rat.Methods Three pair of 21 nucleotide siRNAs directed to CTGF mRNA 483,885 and 946 targets were designed and synthesized by utilizing RNA design software to simulate secondary structure of CTGF mRNA in SD rats. These siRNAs were differently transfected into HSC T6 (an active hepatic stellate cell strain in rat) by oligofectamine package, untreated and transfected unspecific siRNA HSC T6 as control. The expression of CTGF at mRNA level in HSC T6 was determined by reverse transcription polymerase chain reaction (RT PCR).Results Compared with bland controls, the expression of CTGF at mRNA level was markedly down regulated in siRNA transfected HSC T6, degree of down regulation in CTGF mRNA increased in a concentration dependent manner within 50 200nmol/L , especially 483 siRNA 200nmol/L, the expression of CTGF at mRNA level wasn't found to have any significant changes in unspecific siRNA transfected HSC T6. The expression of CTGF at mRNA level down regulated 91%±2%,65%±3%( t = 78.82 , 37.53 , P 0.01 ) and 10%±7%( t = 2.55 , P = 0.06 ), respectively, in 483 siRNA transfected HSC T6 compared with blank control, extent of down regulation in CTGF mRNA decreased in a time dependent manner within 24 72hrs.Conclusions 483 siRNA, which can highly effectively inhibit expression of CTGF gene was screened successfully, its inhibition effect can maintain near 72hrs. It is hopeful that 483 siRNA will be a new nucleic acid drug that can inhibit the development of liver fibrosis through mediating CTGF mRNA degradation.