Establishment of a shRNA-mediated knockdown of NOX2 in human monocytic cell lines

Sammanfattning

Reactive oxygen species (ROS) are widely recognised as signalling molecules that regulate cellular behaviour. Preliminary findings from the Martner lab suggest that ROS generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), which is highly expressed in myeloid cells, may influence tumour progression by modulating tumour-infiltrating macrophage polarisation. However, the effects of stable NOX2 suppression, using RNA interference approaches, remain insufficiently characterised. This thesis aimed to establish a short hairpin RNA (shRNA)-mediated NOX2 knockdown system in human monocytic leukaemia cell lines. To identify a suitable experimental model, HL-60, THP-1 and U937 cells were evaluated for their responsiveness to macrophage-like differentiation. Both HL-60 and THP-1 cells responded to Phorbol-12-myristate-13-acetate (PMA)-induced differentiation; however, only THP-1 cells could efficiently be transfected with siRNA. PMA treatment increased the expression of maturation-associated markers, including gp91phox (NOX2). The efficiency of NOX2-targeting sequences was assessed in THP-1 cells transiently transfected with three different NOX2 targeting siRNAs. Among the tested candidates, siRNA1 and siRNA3 produced the greatest reduction in gp91phox expression and were selected for conversion into shRNA oligonucleotides. To generate a stable knockdown model, shRNA oligonucleotides were annealed and cloned into the pLKO.1 plasmid vector. Although pLKO.1 digestion was successfully optimised, ligation and bacterial transformation did not yield recombinant plasmids. Overall, this thesis established THP-1 cells as a suitable model, optimised conditions for transient siRNA-mediated NOX2 knockdown, identified suitable NOX2-targeting sequences and completed initial steps toward a stable shRNA-mediated knockdown construct, providing a methodological basis for future NOX2-deficient macrophage models and investigation of NOX2-dependent signalling.