Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, 02114, USA.
Department of Ophthalmology, The First Affiliated Hospital of Jinan University, 510632, Guangzhou, China.
Lab Invest. 2019 Dec;99(12):1874-1886. doi: 10.1038/s41374-019-0307-9. Epub 2019 Aug 22.
Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-β2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-β2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-β2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-β2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-β2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-β2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-β2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-β2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.
上皮间质转化 (EMT) 在增生性玻璃体视网膜病变 (PVR) 的发病机制中起着重要作用。我们旨在证明鼠双微体 2 (MDM2) 在转化生长因子-β2 (TGF-β2) 诱导的人视网膜色素上皮细胞 (RPE) EMT 中的作用。免疫荧光法用于评估 PVR 患者眼内膜 (ERM) 中 MDM2 的表达。针对 MDM2 第二个启动子的单指导 (sg)RNA 被克隆到一个突变的慢病毒 Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A 和 H840A) 载体中，用于在 RPE 中表达核酸酶失活 Cas9 (dCas9)/MDM2-sgRNA。此外，MDM2-sgRNA 也被克隆到 pLV-sgRNA-dCas9-Kruppel 相关盒 (KRAB) 载体中，用于表达与转录抑制剂 KRAB 融合的 dCas9/MDM2-sgRNA。通过定量聚合酶链反应 (q-PCR)、western blot 或免疫荧光法评估 TGF-β2 诱导的 MDM2 和 EMT 生物标志物的表达。伤口愈合和增殖实验用于评估 MDM2 在 TGF-β2 诱导的 RPE 反应中的作用。结果发现，MDM2 在 ERM 中明显表达，TGF-β2 诱导 RPE 中 MDM2 和 EMT 生物标志物纤连蛋白、N-钙粘蛋白和波形蛋白的表达。重要的是，我们发现 dCas9/MDM2-sgRNA 阻断了 TGF-β2 诱导的 MDM2 表达和 EMT 生物标志物的表达，而不影响其基础表达，而 dCas9-KRAB/MDM2-sgRNA 则抑制了 RPE 中 MDM2 的基础表达。由于细胞活力大大降低，这些细胞无法持续维持。接下来，我们发现小分子 Nutlin-3 阻断了 MDM2 与 p53 的相互作用，抑制了 TGF-β2 诱导的 RPE 中纤连蛋白和 N-钙粘蛋白的表达，但不抑制波形蛋白的表达，表明 MDM2 在 p53 依赖和非依赖途径中都有功能。最后，我们的实验数据表明，dCas9/MDM2-sgRNA 抑制 TGF-β2 依赖性细胞增殖和迁移，而不干扰未受刺激的基础活性。总之，CRISPR/dCas9 阻断 TGF-β2 诱导的 MDM2 和 EMT 生物标志物表达的能力可用于治疗 PVR。
