Induction of DISE by tumor suppressive microRNAs

Many siRNAs and shRNAs induce a form of cell death in all cancer cells by silencing a set of critical survival genes in a process called death induced by survival gene elimination (DISE). Mechanistically, a 6mer seed sequence (position 2-7 of the guide strand) is sufficient to confer DISE-inducing activity. We have now performed a strand-specific screen testing the toxicity of all 4096 possible 6mer seed sequences in a neutral double-stranded siRNA backbone. We found an asymmetric preference for guanine in positions 1-3 and a G content of >80% of the 6mer seed in the most toxic siRNAs, which target survival genes that are GC rich in their 3'UTR. During over 800 million years of evolution, miRNAs have evolved to avoid guanine in their seed sequences. However, two tumor suppressive miRNAs were found to be killing cancer cells through DISE using G rich toxic 6mer seeds in cells exposed to genotoxic stress: 1) the p53 inducible miR-34 family; and 2) miR-320a, a noncanonical miRNA that is still expressed in cancer cells when miRNA processing genes are mutated. Our data suggest that most miRNAs have evolved to avoid induction of DISE but certain tumor suppressive miRNAs utilize this mechanism to kill cancer cells. Pan Argonaute immunoprecipitation and small RNA cloning in HeyA8 and HCT116 wild-type and Drosha KO cells treated with doxorubicin