Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered 844 lncRNAs that were genetically linked to breast cancer through genome-wide association studies (GWAS). Here, we show that a subset of these lncRNAs alter breast cancer risk by modulating cell proliferation, and provide evidence that a reduced expression on one lncRNA increases breast cancer risk through aberrant DNA replication and repair. We performed pooled CRISPR-Cas13d-based knockdown screens in breast cells to identify which of the 844 breast cancer-associated lncRNAs alter cell proliferation. We selected one of the lncRNAs that increased cell proliferation, KILR, for follow-up functional studies. KILR pull-down followed by mass spectrometry was used to identify binding proteins. Knockdown and overexpression studies were performed to assess the mechanism by which KILR regulates proliferation. We show that KILR functions as a tumor suppressor, safeguarding breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. Mechanistically, KILR sequesters RPA1, a subunit of the RPA complex required for DNA replication and repair. Reduced KILR expression promotes breast cancer cell proliferation by increasing the available pool of RPA1 and speed of DNA replication. Conversely, KILR overexpression promotes apoptosis in breast cancer cells, but not normal breast cells. Our results confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

中文翻译：

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CRISPR-Cas13d 筛选鉴定出 KILR，一种与乳腺癌风险相关的 lncRNA，可调节 DNA 复制和修复


长非编码RNA (lncRNA) 的数量已经超过了蛋白质编码基因，但大多数都没有已知的功能。我们之前通过全基因组关联研究 (GWAS) 发现了 844 个与乳腺癌存在遗传关联的 lncRNA。在这里，我们证明这些 lncRNA 的一个子集通过调节细胞增殖来改变乳腺癌风险，并提供证据表明，一种 lncRNA 表达减少会通过异常的 DNA 复制和修复增加乳腺癌风险。我们在乳腺细胞中进行了基于 CRISPR-Cas13d 的基因敲除筛选，以确定 844 种乳腺癌相关 lncRNA 中哪些会改变细胞增殖。我们选择了一种增加细胞增殖的lncRNA KILR，用于后续功能研究。使用 KILR 下拉和质谱分析来鉴定结合蛋白。进行敲低和过表达研究以评估 KILR 调节增殖的机制。我们证明 KILR 具有肿瘤抑制因子的功能，可保护乳腺细胞免受不受控制的增殖。风险单倍型显着缩短了 KILR 的半衰期，揭示了变体改变癌症风险的另一种机制。从机制上讲，KILR 隔离 RPA1，这是 DNA 复制和修复所需的 RPA 复合物的一个亚基。 KILR 表达减少可通过增加可用的 RPA1 库和 DNA 复制速度来促进乳腺癌细胞增殖。相反，KILR 过度表达会促进乳腺癌细胞凋亡，但不会促进正常乳腺细胞凋亡。 我们的结果证实lncRNA是乳腺癌风险的调节因子，强调在研究GWAS变异时需要注释相关细胞类型中的非编码转录本，并为绘制与lncRNA相关的表型提供可扩展的平台。