Down‐regulation of 3‐Hydroxy‐3‐Methyl Glutaryl Coenzyme‐A Reductase in HEPG2 Cells Using shRNA

Abstract

3‐hydroxy‐3‐methyl glutaryl coenzyme‐A (HMG Co‐A) reductase is an enzyme that catalyzes the committing step in the pathway involved in the de novo biosynthesis of cholesterol in the liver. Pharmacotherapeutic management of hypercholestrolemic patients includes treatment with statins, mevalonate analogs, which are competitive inhibitors of HMG Co‐A reductase. To investigate the effectiveness of short hairpin RNA (shRNA)‐mediated targeted degradation of HMG Co‐A reductase mRNA as a means of inhibition of the expression of the enzyme, plasmid constructs containing sequences from three different regions of the HMG Co‐A mRNA were cloned into pGEM‐3Z vector. Each construct contained 21 nucleotide (nt) sense sequence and the corresponding antisense sequence separated by a 7‐nt spacer sequence. The shRNAs were synthesized by run‐off transcription using T7 RNA polymerase and transfected into human hepatocellular carcinoma (HEPG2) cells. RNA was extracted by NP‐40 lysis method and analyzed by RT‐PCR and Northern blotting. Western blot analysis was performed on whole cell lysates. All three shRNAs caused degradation of the HMG Co‐A mRNA, resulting in the inhibition of the expression of HMG Co‐A reductase, indicating the potential use of these shRNAs to down regulate HMG Co‐A reductase and consequently reduce cholesterol biosynthesis. This work was supported by AMS College of Pharmacy, Long Island University, NY.