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Updated: Aug 13, 2026
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Identification of BRAF Inhibitor Resistance-associated lncRNAs Using Genome-scale CRISPR-Cas9 Transcriptional
Xuan Wen1, Min Han1, Masaki Hosoya1
1Department of Clinical Oncology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Background/Aim:
Approximately 50% of melanomas harbor the BRAF V600E mutation and targeted therapies using BRAF inhibitors improve patient outcomes. Nonetheless, resistance to BRAF inhibitors develops rapidly and remains a challenge in melanoma treatment. In this study, we attempted to isolate long noncoding RNAs (lncRNAs) involved in BRAF inhibitor resistance using a comprehensive screening method.
Materials And Methods:
We used a CRISPR-Cas9 synergistic activation mediator (SAM) protein complex in a genome-scale transcriptional activation assay to screen for candidate lncRNA genes related to BRAF inhibitor resistance. Correlation analysis was performed between expression levels of isolated lncRNA genes and IC50 of dabrafenib in a BRAF-mutated melanoma cell line. Next, online databases were used to construct the lncRNA-miRNA-mRNA regulatory network. Finally, we evaluated the significance of the expression levels of these lncRNAs and mRNAs as biomarkers using clinical specimens.
Results:
We isolated three BRAF inhibitor resistance-associated lncRNA genes, namely SNHG16, NDUFV2-AS1, and LINC01502. We constructed a lncRNA-miRNA-mRNA network of 13 nodes consisting of three lncRNAs, six miRNAs, and four mRNAs. The lncRNAs and target mRNAs from each regulatory axis significantly and positively correlated with each other. Finally, Kaplan-Meier analysis showed that higher expression levels of MITF, which was up-regulated by LINC01502, were significantly associated with worse prognosis in BRAF V600E-mutated melanoma.
Conclusion:
The identification of these BRAF inhibitor resistance-associated lncRNA genes at the genomic scale and the establishment of the lncRNA-miRNA-mRNA regulatory network provides new insights into the underlying mechanisms of BRAF inhibitor resistance in melanoma.
Insights
Researchers identified three long noncoding RNAs (lncRNAs) linked to BRAF inhibitor resistance in melanoma. These lncRNAs, along with microRNAs and mRNAs, form a network that may drive resistance and affect patient prognosis.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- BRAF V600E mutation is present in ~50% of melanomas.
- BRAF inhibitors improve outcomes but resistance is a major challenge.
- Understanding resistance mechanisms is crucial for effective melanoma treatment.
Purpose of the Study:
- To identify long noncoding RNAs (lncRNAs) associated with BRAF inhibitor resistance in melanoma.
- To elucidate the regulatory network involving lncRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in resistance.
- To evaluate the potential of identified genes as biomarkers for melanoma prognosis.
Main Methods:
- Genome-scale transcriptional activation assay using CRISPR-Cas9 SAM complex to screen for lncRNAs.
- Correlation analysis of lncRNA expression and drug resistance (IC50).
- Construction of a lncRNA-miRNA-mRNA regulatory network and validation in clinical specimens.
Main Results:
- Three BRAF inhibitor resistance-associated lncRNAs identified: SNHG16, NDUFV2-AS1, and LINC01502.
- A 13-node lncRNA-miRNA-mRNA network was constructed.
- Higher LINC01502 expression correlated with increased MITF levels and worse prognosis in BRAF V600E melanoma patients.
Conclusions:
- Genomic screening identified key lncRNAs involved in BRAF inhibitor resistance.
- The established regulatory network provides insights into resistance mechanisms.
- Identified lncRNAs and associated mRNAs may serve as prognostic biomarkers for melanoma.
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