Pharmacological versus genetic inhibition of heme oxygenase-1 – the comparison of metalloporphyrins, shRNA and CRISPR/Cas9 system

Olga Mucha; Paulina Podkalicka; Maria Czarnek; Anna Biela; Mateusz Mieczkowski; Neli Kachamakova-Trojanowska; Jacek Stępniewski; Alicja Józkowicz; Józef Dulak; Agnieszka Łoboda

doi:10.18388/abp.2017_2542

Olga Mucha
Paulina Podkalicka
Maria Czarnek
Anna Biela
Mateusz Mieczkowski
Neli Kachamakova-Trojanowska
Jacek Stępniewski
Alicja Józkowicz
Józef Dulak
Agnieszka Łoboda

DOI: https://doi.org/10.18388/abp.2017_2542

Abstract

Inhibition of heme oxygenase-1 (HO-1, encoded by HMOX1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, may be a valuable therapy in various pathophysiological processes, including tumorigenesis. We compared the effect of chemical inhibitors – metalloporphyrins, with genetic tools - shRNA and CRISPR/Cas9 systems, to knock-down (KD)/knock-out (KO) HO-1 expression/activity. 293T cells were incubated with metalloporphyrins, tin and zinc protoporphyrins (SnPPIX and ZnPPIX, respectively) or were either transduced with lentiviral vectors encoding different shRNA sequences against HO-1 or were modified by CRISPR/Cas9 system targeting HMOX1. Metalloporphyrins decreased HO activity but concomitantly strongly induced HO-1 mRNA and protein in 293T cells. On the other hand, basal and hemin-induced HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA, protein and activity level. Nevertheless, silencing effect was much stronger when CRISPR/Cas9-mediated knock-out was performed. Most of the clones harboring mutations within HMOX1 locus did not express HO-1 and failed to increase bilirubin concentration after hemin stimulation. Furthermore, CRISPR/Cas9-mediated HO-1 depletion decreased 293T viability, growth, clonogenic potential and increased sensitivity to H₂O₂ treatment. In summary, we have shown that all technologies can be used for inhibition of HO activity in vitro; however, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach.

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