RNAi Knockdown of 11β‐Hydroxysteroid Dehydrogenase‐1 in GH4C1 Rat Pituitary Cells

The enzyme 11β‐hydroxysteroid dehydrogenase‐1 (11β‐HSD‐1) is thought to play a critical role in the regulation of intracellular cortisol (human) and corticosterone (rat). 11β‐HSD‐1 is found in several cell types including pituitary, adipose, and liver. Increased message and/or activity are characteristic of both human and animal models of obesity. The purpose of this study was to identify the effectiveness of three RNAi sequences in suppressing 11β‐HSD‐1 message. The RNAi sequences (designated A, B, and C) were purchased from Invitrogen and used to transfect cultured GH4C1 rat pituitary cells. RNAi‐transfected and control cells were harvested at 24, 48, 72, and 144 hours after transfection. Total RNA was extracted and treated to remove contaminating DNA. cDNA templates were then made from 500 ng RNA via reverse transcriptase reactions. qRT‐PCR was performed to measure mRNA levels of 11β‐HSD‐1 and β‐actin, which served as controls. Two of the three sequences (A, C) suppressed 11β‐HSD‐1 message by approximately 90% at both 24 and 48 hours post‐transfection. Message suppression had decreased to nearly 50% by 144 hours post‐transfection. To our knowledge this is the first knockdown of 11β‐HSD‐1 using RNAi. Future investigations using these RNAi fragments in other cell types will contribute to our knowledge of how this enzyme plays a role in the regulation of intracellular glucocorticoids.