Mucha Olga, Podkalicka Paulina, Czarnek Maria, Biela Anna, Mieczkowski Mateusz, Kachamakova-Trojanowska Neli, Stepniewski Jacek, Jozkowicz Alicja, Dulak Jozef, Loboda Agnieszka
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
Acta Biochim Pol. 2018;65(2):277-286. doi: 10.18388/abp.2017_2542. Epub 2018 Apr 25.
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 HO 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.
血红素加氧酶-1（HO-1，由HMOX1编码）是一种具有细胞保护、抗凋亡和抗炎作用的酶，抑制该酶可能在包括肿瘤发生在内的各种病理生理过程中成为一种有价值的治疗方法。我们比较了化学抑制剂——金属卟啉与基因工具——shRNA和CRISPR/Cas9系统对HO-1表达/活性的敲低（KD）/敲除（KO）效果。将293T细胞与金属卟啉、锡原卟啉和锌原卟啉（分别为SnPPIX和ZnPPIX）孵育，或者用编码针对HO-1的不同shRNA序列的慢病毒载体进行转导，或者用靶向HMOX1的CRISPR/Cas9系统进行修饰。金属卟啉降低了HO活性，但同时强烈诱导了293T细胞中HO-1的mRNA和蛋白质表达。另一方面，在shRNA KD 293T细胞系中，仅在mRNA和蛋白质水平上证实了对HO-1有轻微的基础抑制作用，对酶活性无显著影响。然而，当进行CRISPR/Cas9介导的敲除时，沉默效果要强得多。大多数在HMOX1基因座内携带突变的克隆不表达HO-1蛋白，并且在血红素刺激后未能增加胆红素浓度。此外，CRISPR/Cas9介导的HO-1缺失降低了293T细胞的活力、生长、克隆形成潜力，并增加了对HO处理的敏感性。总之，我们已经表明，并非所有技术都能以相同效率用于体外抑制HO活性。在我们的研究中，使用基因工具，尤其是CRISPR/Cas9方法，可以获得最有效和最易理解的结果。
