AbstractRecently, both 5p and 3p miRNA strands are being recognized as functional instead of only one, leaving many miRNA strands uninvestigated. To determine whether both miRNA strands, which have different mRNA-targeting sequences, cooperate to regulate pathways/functions across cancer types, we evaluate genomic, epigenetic, and molecular profiles of >5200 patient samples from 14 different cancers, and RNA interference and CRISPR screens in 290 cancer cell lines. We identify concordantly dysregulated miRNA 5p/3p pairs that coordinately modulate oncogenic pathways and/or cell survival/growth across cancers. Down-regulation of both strands of miR-30a and miR-145 recurrently increased cell cycle pathway genes and significantly reduced patient survival in multiple cancers. Forced expression of all four strands show cooperativity, reducing cell cycle pathways and inhibiting lung cancer cell proliferation and migration. Therefore, we identify miRNA whose 5p/3p strands function together to regulate core tumorigenic processes/pathways and reveal a previously unknown pan-cancer miRNA signature with patient prognostic power.