Abstract
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 KrasG12D/Trp53L/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.
Highlights
Lung cancer is one of the most devastating diseases worldwide, accounting for about 24% of cancer-related death annually [1]
Such technique provides the base for large scale screening of tumor suppressors in genetically engineered mouse model (GEMM)
In vivo miRNA knockout screening identifies tumor suppressors in lung tumorigenesis To identify tumor-suppressive miRNAs that are down-regulated in lung tumorigenesis, we first generated a list of miRNA candidates based on bioinformatic analyses of miRNA expression and TNM classification from the TCGA database
Summary
Lung cancer is one of the most devastating diseases worldwide, accounting for about 24% of cancer-related death annually [1]. MiRNAs participate in almost all the cellular activities, including apoptosis, cell proliferation, invasion, metastasis, and angiogenesis [4]. MiR-30b functions as a tumor suppressor through the inhibition of cell proliferation, metastasis and epithelial-to-mesenchymal transition in different carcinomas [5,6,7,8]. The miR-146a is down-regulated in lung cancer and frequently associates with distant organ metastasis and poor prognosis [9, 10]. It’s worth noting that most of these studies have been done in vitro and/or in cell lines. Systematic identification of those important miRNAs using in vivo screening system remains an obstacle to gain deep understanding of the miRNAs’ function in lung tumorigenesis
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