Gene silencing of human RAMP2 mediated by short-interfering RNA

Adrenomedullin (AM) is a regulatory peptide widely expressed, along its receptors, in cells and tissues, of which it controls many basic and specific functions acting in an autocrine-paracrine manner. However, the unequivocal demonstration of the physiological relevance of the regulatory role of AM would require the study of cells where the endogenous AM system has been suppressed. For this task we developed a technique to silence the AM gene in human umbilical vein endothelial cells (HUVECs) and the human embryonal kidney cell line (HEK-293). AM acts via two subtypes of receptor, named AM1 and AM2, which derive from the interaction of the calcitonin receptor-like receptors with two chaperones, called receptor activity modifying proteins (RAMP2 and RAMP3). Hence, we developed a protocol to suppress the human AM1 receptor by silencing the RAMP2 gene by transfection with short interfering RNAs (siRNAs). HUVECs were transfected using a new Ambion transfection reagent. RAMP2 gene silencing was determined in HUVECs by measuring RAMP2 mRNA levels in transfected and control cells by real-time polymerase chain reaction. The RAMP2 gene silencing was approximately 60% and was observed 48 h after transfection. Matrigel assay in vitro was carried out to evaluate the effects of siRNA sequences. HUVECs cells were plated on matrigel and the analysis of capillary-like tubule formation showed that the cells were viable. The knockdown of the RAMP2 gene decreased the formation of tubes in response to 10(-8) M AM. The conclusion is drawn that siRNA technology can be successfully used in the investigations on AM and AM receptor functions.