shRNAs targeting mouse Adam10 diminish cell response to proinflammatory stimuli independently of Adam10 silencing

RNA interference is one of the common methods of studying protein functions. In recent years critical reports have emerged indicating that off-target effects may have a much greater impact on RNAi-based analysis than previously assumed. We studied the influence of Adam10 and Adam17 silencing on MC38CEA cell response to proinflammatory stimuli. Eight lentiviral vector-encoded shRNAs that reduced ADAM10 expression, including two that are specific towards ADAM17, caused inhibition of cytokine-induced Nos2 expression presumably via off-target effects. ADAM10 silencing was not responsible for this effect because: (i) CRISPR/Cas9 knockdown of ADAM10 did not affect Nos2 levels; (ii) ADAM10 inhibitor increased rather than decreased Nos2 expression; (iii) overexpression of ADAM10 in the cells with shRNA-silenced Adam10 did not reverse the effect induced by shRNA; (iv) shRNA targeting ADAM10 resulted in decrease of Nos2 expression even in ADAM10-deficient cells. The studied shRNAs influenced transcription of Nos2 rather than stability of Nos2 mRNA. They also affected stimulation of Ccl2 and Ccl7 expression. Additionally, we used vectors with doxycycline-inducible expression of chosen shRNAs and observed reduced activation of NF-κB and, to a lesser extent, AP-1 transcription factors. We discuss the requirements of strict controls and verification of results with complementary methods for reliable conclusions of shRNA-based experiments.