The oncogenic microRNA miR-222 promotes human LINE-1 retrotransposition

The Long Interspersed Element-1 (LINE-1) contributes significantly to carcinogenesis and to tumor heterogeneity in many cancer types, including hepatocellular carcinoma (HCC), by its autonomous retrotransposition (RTP) and by its ability to retrotranspose some non-autonomous transposable elements. Previously, multiple proteins and a few microRNAs (miRs) were described as regulators of LINE-1 RTP. Here, we demonstrate that miR-222, which is oncogenic in HCC, promotes LINE-1 RTP in human HCC and some other cell lines in vitro, and that both miR-222-3p and miR-222-5p activate LINE-1 RTP in a cell type-specific manner. We generated miR-222-knockout mutants of the Huh7 and FLC4 HCC cell lines, and performed RNA-seq analysis of Huh7/miR-222-knockout cells and global proteomics analysis of both Huh7 and FLC4 miR-222-knockout mutants. We demonstrate that miR-222 decreases let-7c expression in both Huh7 and FLC4 cells, and that this decrease contributes to promotion of LINE-1 RTP by miR-222 in Huh7 cells. Overall design: RNA-seq was performed on the stable single clones of Huh7 cellsgenerated using the CRISPR-Cas9 system: Huh7/Cas9/miR-222-KO versus similar control clones expressing a control gRNA that has no targets in the human genome (four clones in each group).