In-vitro analysis of siRNA based inhibition of function of activated protein C (APC): Towards an alternative therapeutic option for hemophilia.
D. Mirgal1, V. Pujari1, K. Ghosh2, S. Shetty1 (1Mumbai, Maharashtra, 2Surat,Gujrath, India)
Bleeding disorders, coagulation and fibrinolytic factors
Time: 17:15 - 18:15
Objective: To analyze different siRNA sequences directed against different functional domains of protein C gene (PROC) and study their silencing effect on protein C in an in vitro and in vivo system using human hepatic cell lines and mice respectively, which may serve as an alternate therapeutics for hemophilia management.
Methods: Human HepG2 hepatic carcinoma cells were cultured and reverse transfected using various concentrations and different combinations of different siRNA’s specific to PROC, in presence of lipofectamine, to down regulate the PROC gene expression. After successful transfections of particular siRNA, RNA was isolated from the transfected cells and was converted to cDNA using reverse transcriptase followed by PCR amplification using real time PCR. PROC down regulation was determined using qRT-PCR by calculating fold change.
Results: The individual siRNA against; - Exon 3 of PROC showed 35% of gene silencing at 10pmol concentration and 75% silencing with 12pmoL concentration. - Exon 9 of PROC showed 25% of gene silencing at the concentration of 3pmol and 70% silencing at 6pmol concentration. - Exon 7 of PROC showed 50% of gene silencing at the concentration of 6pmol and 80% silencing at 10pmol concentration. The combination of siRNA against; - Exon 3 and exon 9 of PROC showed 27 % of gene silencing even at 1.5pmol concentration and 67% silencing at 3pmol concentration. - Exon 9 and exon 7 of PROC showed 36% of gene silencing even at 3pmol concentration and 59% silencing at 4.5pmol concentration. - Exon 3 and exon 7 of PROC showed 34% of gene silencing even at 1.5pmol concentration and 58% silencing at 3pmol concentration. This shows that siRNA targeting different exons of protein C gene in combinations gives higher silencing effect at lower concentration than individually.
Conclusion: These in-vitro findings show the feasibility of silencing PROC by a siRNA based approach as an alternate therapeutic approach for hemophilia.