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

• Authors: Olga Mucha , Paulina Podkalicka , Maria Czarnek , Anna Biela , Mateusz Mieczkowski , Neli Kachamakova-Trojanowska , Jacek Stepniewski , Alicja Jozkowicz , Jozef Dulak , Agnieszka Loboda
• Languages of publication: EN

Abstracts

EN

Inhibition of heme oxygenase-1 (HO-1, encoded by HMOX1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, may serve as 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, only slight basal HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA and protein level with no significant effect on enzyme activity. 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 protein 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 not all technologies can be used for inhibition of HO activity in vitro with the same efficiency. In our hands, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach.

Keywords

EN

CRISPR/Cas9; shRNA; inhibitors; heme oxygenase-1; HO-1; off-target

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2018
• Volume: 65
• Issue: 2
• Pages: 277-286

Dates

published

2018

received

2017-12-08

revised

2018-01-19

accepted

2018-03-07

(unknown)

2018-04-25

Contributors

author

Olga Mucha

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Paulina Podkalicka

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Maria Czarnek

• Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Anna Biela

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Mateusz Mieczkowski

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Neli Kachamakova-Trojanowska

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Jacek Stepniewski

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Alicja Jozkowicz

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

author

Jozef Dulak

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
• Kardio-Med Silesia, Zabrze, Poland

author

Agnieszka Loboda

• Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

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Identifiers

DOI

10.18388/abp.2017_2542