In vivo miRNA knockout screening identifies miR-190b as a novel tumor suppressor

Author summary CRISPR/Cas9 has been widely used for genomic editing, especially for in vivo somatic gene mutation/fusion engineering in cancer research. Such technique provides the base for large scale screening of tumor suppressors in genetically engineered mouse model (GEMM). Up to date, no effective miRNA somatic knockout system has been reported yet, mainly ascribed to the imperfect matching mechanism for miRNA functional execution. Here we develop the dual guide RNA (dgRNA)-mediated CRISPR/Cas9 knockout system for the effective miRNA knockout in Kras(G12D)Trp53(L/L) (KP) lung cancer mouse model. Through screening of 16 miRNAs down-regulated in human lung cancer, we identify multiple tumor-suppressive miRNAs: two previously reported miRNAs including miR-30b and miR-146a, and one novel miRNA miR-190b. We further demonstrate that miR-190b functions through targeting the Hus1 gene to inhibit lung tumorigenesis. Taken together, our study develops the first in vivo miRNA knockout platform for functionally screening of tumor-suppressive miRNAs in GEMM and identifies the miR-190b as a new tumor suppressor in lung tumorigenesis. MicroRNAs (miRNAs) play important roles in the development of various cancers including lung cancer which is one of the devastating diseases worldwide. How miRNAs function in de novo lung tumorigenesis remains largely unknown. We here developed a CRISPR/Cas9-mediated dual guide RNA (dgRNA) system to knockout miRNAs in genetically engineered mouse model (GEMM). Through bioinformatic analyses of human lung cancer miRNA database, we identified 16 downregulated miRNAs associated with malignant progression and performed individual knockout with dgRNA system in Kras(G12D)/Trp53(L/L) (KP) mouse model. Using this in vivo knockout screening, we identified miR-30b and miR-146a, which has been previously reported as tumor suppressors and miR-190b, a new tumor-suppressive miRNA in lung cancer development. Over-expression of miR-190b in KP model as well as human lung cancer cell lines significantly suppressed malignant progression. We further found that miR-190b targeted the Hus1 gene and knockout of Hus1 in KP model dramatically suppressed lung tumorigenesis. Collectively, our study developed an in vivo miRNA knockout platform for functionally screening in GEMM and identified miR-190b as a new tumor suppressor in lung cancer.