Open AccessshRNAs targeting mouseAdam10diminish cell response to proinflammatory stimuli independently ofAdam10silencing
Author(s) -
Maria Czarnek,
Krystyna Stalińska,
Katarzyna Sarad,
Joanna Bereta
Publication year - 2022
Publication title -
biology open
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.936
H-Index - 41
ISSN - 2046-6390
DOI - 10.1242/bio.059092
Subject(s) - small hairpin rna , gene knockdown , adam10 , gene silencing , rna interference , biology , proinflammatory cytokine , microbiology and biotechnology , cancer research , rna , cell culture , immunology , inflammation , genetics , disintegrin , gene , metalloproteinase , matrix metalloproteinase
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.