A plant immune protein enables broad antitumor response by rescuing microRNA deficiency in cancers [mRNA-seq]

Cancer cells are featured with uncontrollable activation of cell cycle and proliferation,and microRNA deficiency drives tumorigenesis. The RNA-dependent RNA polymerase (RDR) is essential for small RNA-mediated immune response in plants, but absent in vertebrates with adaptive immunity. Here we show that ectopic expression of plant RDR1 can generally inhibit the proliferation of different cancer cells. Interestingly, we find that abnormal AGO2-free microRNA isoforms with 1-nucleotide shortening at the 3' end are widely accumulated in many tumors. RDR1 with nucleotidyltransferase activity can recognize and modify these problematic microRNAs with mononucleotides to restore their AGO2 loading efficiency, which eventually rescues microRNA deficiency and elevates microRNA expression to target cell cycle in cancer specifically. The wide antitumor effects of RDR1 can be visualized in multiple xenograft tumor models in vivo, which can be realized by adeno-associated virus-mediated delivery. Altogether, we develop a broad microRNA-mediated antitumor stratagem using the plant immune protein RDR1. Overall design: Seven solid tumors (A549, HCT116, HepG2, HeLa, H1299, PC-3 and U-2 OS), three leukemia (Jurkat, K562 and NALM6), and three non-cancer somatic cell lines (NIH/3T3, RPE-1 and WPMY-1), as well as two embryonic stem cell lines (mouse V6.5 and human WIBR3) with or without RDR1 expression were used for RNA-seq at different time points. Small RNA-seq were performed at A549, HCT116, HepG2, HeLa, H1299, Jurkat, NIH/3T3 and RPE-1 cell lines with or without RDR1 expression for 5 or 6 days. siRNA-mediated AGO2 knockdown in cancer A549 cells with or without RDR1 expression were used for small RNA-seq to analyze the tailing percentage of miRNAs (siNC, Negative control). AGO2-IP followed by small RNA-seq analysis was performed from the A549 cell lines and HepG2 cell lines with inducible RDR1 expression (including miR-34c transfection assay). Tumor samples from the A549, H1299, PC-3, Jurkat and NALM-6 xenograft models were used for RNA-seq analysis (including AAV-mediated RDR1 delivery in A549 xenografts). EGFP+ cells in the peripheral blood of Jurkat xenotransplantation mice were sorted from vector group or AtRDR1 group at different time and used for single-cell RNA-seq. For nanoparticle-mediated RDR1 delivery, A549, HeLa, H1299, NIH/3T3 and RPE-1 cells were transfected with RDR1-containing nanoparticles and used for RNA-seq.