Department of Medical Oncology, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, USA.
Department of Molecular Medicine, City of Hope Comprehensive Cancer Center and National Medical Center, Duarte, CA, USA.
Carcinogenesis. 2021 Feb 11;42(1):48-57. doi: 10.1093/carcin/bgaa048.
Breast cancer (BC) remains one of the major causes of cancer deaths in women. Over half of all BCs carry genetic defects in the gene encoding p53, a powerful tumor suppressor. P53 is known as the 'guardian of the genome' because it is essential for regulating cell division and preventing tumor formation. Ral-interacting protein (RLIP) is a modular protein capable of participating in many cellular functions. Blocking this stress-responsive protein, which is overexpressed during malignancy, enables BC cells to overcome the deleterious effects of p53 loss more effectively. In the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system, a single-guide RNA (sgRNA) recognizes a specific DNA sequence and directs the endonuclease Cas9 to make a double-strand break, which enables editing of targeted genes. Here, we harnessed CRISPR/Cas9 technology to target the RLIP gene in BC cells. We screened sgRNAs using a reporter system and lentivirally delivered them, along with Cas9, to BC cells for validation. We then assessed the survival, proliferation, and tumorigenicity of BC cells in vitro and the growth of tumors in vivo after CRISPR-mediated knockdown of RLIP. Doxycycline-inducible expression of Cas9 in BC cells transduced with lentiviral vectors encoding the sgRNAs disrupted the RLIP gene, leading to inhibition of BC cell proliferation both in vitro and in vivo, with resected tumors showing reduced levels of the survival and proliferation markers Ki67, RLIP, pAkt, and survivin, the cell cycle protein CDK4, and the mesenchymal marker vimentin, as well as elevated levels of the differentiation protein E-cadherin and pro-apoptotic protein Bim. Inducible Cas9/sgRNA-transduced BC cells without doxycycline treatment did not exhibit altered cell survival or proliferation in vitro or in vivo. Our study provides proof-of-concept that the CRISPR/Cas9 system can be utilized to target RLIP in vitro and in vivo.
乳腺癌（BC）仍然是女性癌症死亡的主要原因之一。超过一半的 BC 携带编码 p53 的基因的遗传缺陷，p53 是一种强大的肿瘤抑制因子。p53 被称为“基因组的守护者”，因为它对于调节细胞分裂和防止肿瘤形成至关重要。Ral 相互作用蛋白（RLIP）是一种模块化蛋白，能够参与许多细胞功能。阻断这种在恶性肿瘤中过度表达的应激反应蛋白，可以使 BC 细胞更有效地克服 p53 缺失的有害影响。在成簇规律间隔短回文重复/CRISPR 相关蛋白（CRISPR/Cas9）系统中，单个向导 RNA（sgRNA）识别特定的 DNA 序列，并指导内切酶 Cas9 产生双链断裂，从而实现靶向基因的编辑。在这里，我们利用 CRISPR/Cas9 技术靶向 BC 细胞中的 RLIP 基因。我们使用报告基因系统筛选 sgRNA，并通过慢病毒将它们与 Cas9 一起递送到 BC 细胞中进行验证。然后，我们评估了 CRISPR 介导的 RLIP 敲低后 BC 细胞在体外的存活、增殖和致瘤性，以及体内肿瘤的生长。转导带有编码 sgRNA 的慢病毒载体的 BC 细胞中 Cas9 的诱导表达破坏了 RLIP 基因，导致 BC 细胞在体外和体内的增殖均受到抑制，切除的肿瘤显示出存活和增殖标志物 Ki67、RLIP、pAkt 和 survivin、细胞周期蛋白 CDK4 和间充质标志物波形蛋白的水平降低，以及分化蛋白 E-钙粘蛋白和促凋亡蛋白 Bim 的水平升高。没有使用强力霉素处理的诱导型 Cas9/sgRNA 转导的 BC 细胞在体外或体内均未显示出细胞存活或增殖的改变。我们的研究提供了概念验证，证明 CRISPR/Cas9 系统可用于体外和体内靶向 RLIP。
