Abstract：
OBJECTIVE: 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.
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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
目的：大约50%的黑色素瘤携带BRAFV600E突变，使用BRAF抑制剂的靶向治疗可改善患者预后。尽管如此，对BRAF抑制剂的耐药性发展迅速，在黑色素瘤治疗中仍然是一个挑战。在这项研究中,我们尝试使用综合筛选方法分离与BRAF抑制剂耐药相关的长链非编码RNA(lncRNA).
方法：我们在基因组规模转录激活试验中使用CRISPR-Cas9协同激活介质（SAM）蛋白复合物来筛选与BRAF抑制剂抗性相关的lncRNA候选基因。在BRAF突变的黑色素瘤细胞系中分离的lncRNA基因的表达水平与dabrafenib的IC50之间进行相关性分析。接下来,在线数据库用于构建lncRNA-miRNA-mRNA调控网络。最后,我们使用临床样本评估了这些lncRNAs和mRNAs作为生物标志物的表达水平的意义.
结果：我们分离了三个BRAF抑制剂抗性相关的lncRNA基因，即SNHG16、NDUFV2-AS1和LINC01502。我们构建了一个由13个节点组成的lncRNA-miRNA-mRNA网络，六个miRNA，和四个mRNA。来自每个调控轴的lncRNA和靶mRNA彼此显著且正相关。最后,Kaplan-Meier分析显示MITF表达水平较高,在BRAFV600E突变的黑色素瘤中，LINC01502上调与预后较差显着相关。
结论：在基因组规模上鉴定这些BRAF抑制剂抗性相关的lncRNA基因，以及lncRNA-miRNA-mRNA调控网络的建立，为黑色素瘤中BRAF抑制剂抗性的潜在机制提供了新的见解。
方法：我们在基因组规模转录激活试验中使用CRISPR-Cas9协同激活介质（SAM）蛋白复合物来筛选与BRAF抑制剂抗性相关的lncRNA候选基因。在BRAF突变的黑色素瘤细胞系中分离的lncRNA基因的表达水平与dabrafenib的IC50之间进行相关性分析。接下来,在线数据库用于构建lncRNA-miRNA-mRNA调控网络。最后,我们使用临床样本评估了这些lncRNAs和mRNAs作为生物标志物的表达水平的意义.
结果：我们分离了三个BRAF抑制剂抗性相关的lncRNA基因，即SNHG16、NDUFV2-AS1和LINC01502。我们构建了一个由13个节点组成的lncRNA-miRNA-mRNA网络，六个miRNA，和四个mRNA。来自每个调控轴的lncRNA和靶mRNA彼此显著且正相关。最后,Kaplan-Meier分析显示MITF表达水平较高,在BRAFV600E突变的黑色素瘤中，LINC01502上调与预后较差显着相关。
结论：在基因组规模上鉴定这些BRAF抑制剂抗性相关的lncRNA基因，以及lncRNA-miRNA-mRNA调控网络的建立，为黑色素瘤中BRAF抑制剂抗性的潜在机制提供了新的见解。
