Knockdown Of TRPC4 Expression In Human Corneal Epithelial Cells Suppresses Proliferation

Abstract

Abstract: : Purpose:Canonical transient receptor potential protein isoforms (TRPC 1–7) are components of receptor and store operated channels (SOC). In different homotypic and heterotypic combinations, they play fundamental roles in intracellular Ca²⁺ homeostasis. Our previous studies suggest that TRPC4 is plasma membrane delimited and needed for the mitogenic response to epidermal growth factor (EGF). To gain insight about the functional importance of TRP4 expression, we knocked down TRP4 expression in human corneal epithelial cells (HCEC). Methods: Four different siRNAs specific for TRPC4 were constructed using Silencer siRNA construction kit (Ambion). The siRNAs were transfected into HCE cells using Oligofectamine ® (Invitrogen). At 72 hours of post–transfection, RT–PCR and immunoblot analysis were performed to test the knockdown of TRP4 mRNA and protein expression using specific TRPC4 primers and antibody respectively. The mitogenic response to EGF (5 ng/ml) in TRPC4 knockdown HCEC was evaluated based on measurements of [³H] thymidine incorporation. Results: Transfected HCE cell viability was uncompromised following siRNA transfection. Immunoblot analysis showed that two of the four synthesized TRPC4 siRNA (i.e siRNA2 and 3) effectively decreased TRPC4 protein expression by 85 and 89% relative to the control respectively. RT–PCR showed that siRNA decreased TRPC4 mRNA expression by 88%. The mitogenic response to EGF in transfected HCE cells using either siRNA2 or siRNA3 was reduced by 54 and 58% relative to nontransfected counterpart, respectively. Conclusions:In HCE cells, TRPC4 siRNA specifically and effectively reduced both TRP4 mRNA and protein expression without compromising cell viability. The suppressed mitogenic response to EGF following TRPC4 knockdown suggests that this isoform is important for SOC functional activity.

Keywords: cornea: epithelium • growth factors/growth factor receptors • gene/expression