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MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs.

ABSTRACT: MicroRNAs (miRNAs) are post-transcriptional gene regulators that play important roles in the control of cell fitness, differentiation, and development. The CRISPR-Cas9 gene-editing system is composed of the Cas9 nuclease in complex with a single guide RNA (sgRNA) and directs DNA cleavage at a predetermined site. Several CRISPR-Cas9 libraries have been constructed for genome-scale knockout screens of protein function; however, few libraries have included miRNA genes. Here we constructed a miRNA-focused CRISPR-Cas9 library that targets 1594 (85%) annotated human miRNA stem-loops. The sgRNAs in our LX-miR library are designed to have high on-target and low off-target activity, and each miRNA is targeted by four to five sgRNAs. We used this sgRNA library to screen for miRNAs that affect cell fitness of HeLa or NCI-N87 cells by monitoring the change in frequency of each sgRNA over time. By considering the expression in the tested cells and the dysregulation of the miRNAs in cancer specimens, we identified five HeLa pro-fitness and cervical cancer up-regulated miRNAs (miR-31-5p, miR-92b-3p, miR-146b-5p, miR-151a-3p, and miR-194-5p). Similarly, we identified six NCI-N87 pro-fitness and gastric cancer up-regulated miRNAs (miR-95-3p, miR-181a-5p, miR-188-5p, miR-196b-5p, miR-584-5p, and miR-1304-3p), as well as three anti-fitness and down-regulated miRNAs (let-7a-3p, miR-100-5p, and miR-149-5p). Some of those miRNAs are known to be oncogenic or tumor-suppressive, but others are novel. Taken together, the LX-miR library is useful for genome-wide unbiased screening to identify miRNAs important for cellular fitness and likely to be useful for other functional screens.

SUBMITTER: Kurata JS

PROVIDER: S-EPMC6004052 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs.

Kurata Jessica S JS Lin Ren-Jang RJ

RNA (New York, N.Y.) 20180502 7

MicroRNAs (miRNAs) are post-transcriptional gene regulators that play important roles in the control of cell fitness, differentiation, and development. The CRISPR-Cas9 gene-editing system is composed of the Cas9 nuclease in complex with a single guide RNA (sgRNA) and directs DNA cleavage at a predetermined site. Several CRISPR-Cas9 libraries have been constructed for genome-scale knockout screens of protein function; however, few libraries have included miRNA genes. Here we constructed a miRNA-f ...[more]

PMID: 29720387

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Project description:BackgroundThe development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance.MethodsGenome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging.ResultsWe found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors.ConclusionsThrough systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.

Dataset Information

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs.

Publications

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs.

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