Cellular knock-down of quinone reductase 2: a laborious road to successful inhibition by RNA interference |
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Authors: | Chomarat Pascale Cogé Francis Guénin Sophie P Mailliet François Vella Fanny Mallet Catherine Giraudet Stéphanie Nagel Nadine Leonce Stéphane Ferry Gilles Delagrange Philippe Boutin Jean A |
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Institution: | Pharmacologie Moléculaire et Cellulaire, Institut de Recherches SERVIER, 125 chemin de Ronde, 78290 Croissy-sur-Seine, France. |
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Abstract: | NRH:quinone oxidoreductase 2 (QR2) is a long forgotten oxidoreductive enzyme that metabolizes quinones and binds melatonin. We used the potency of the RNA interference (RNAi)-mediated gene silencing to build a cellular model in which the role of QR2 could be studied. Because standard approaches were poorly successful, we successively used: (1) two chemically synthesized fluorescent small interfering RNA (siRNA) duplexes designed and tested for their gene silencing capacity leading to a maximal 40% QR2 gene silencing 48h post-transfection; (2) double transfection and cell-sorting of high fluorescent siRNA-transfected HT22 cells further enhancing QR2 RNAi silencing to 88%; (3) stable QR2 knock-down HT22 cell lines established with H1and U6 promoter driven QR2 short hairpin RNA (shRNA) encoding vectors, resulting in a 71-80% reduction of QR2 enzymatic activity in both QR2 shRNA HT22 cells. Finally, as a first step in the study of this cellular model, we observed a 42-48% reduction of menadione/BNAH-mediated toxicity in QR2 shRNA cells compared to the wild-type HT22 cells. Although becoming widespread and in some cases effective, siRNA-mediated cellular knock-down proves in the present work to be of marginal efficiency. Much development is required for this technique to be of general application. |
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Keywords: | Quinone reductase 2 RNA interference Short hairpin RNA HT22 Transfection |
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