Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells
Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells
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DOI：
10.1016/j.exer.2019.107716
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发表时间：
2019-09-01
影响因子：
3.4
通讯作者：
Lei, Hetian
中科院分区：
文献类型：
--
作者：
Chen, Na;Hu, Zhengping;Lei, Hetian
小鼠双微体（MDM）2第二启动子中的单核苷酸多态性（SNP）309 G等位基因增强了Mdm 2的玻璃体诱导表达和肿瘤抑制蛋白p53的降解。这种MDM 2（SNP 309 G）有助于某些癌症的发展和实验性增殖性玻璃体视网膜病变。本研究的目的是发现一种新的策略，仅阻断玻璃体诱导的Mdm 2表达，以防止玻璃体诱导的细胞增殖和存活，从而找到一种潜在的治疗增殖相关疾病的新策略。我们在核酸酶结构域（分别为RuvCl和HNH）内的化脓链球菌（Sp）Cas9中产生了两个突变（D10 A和H840 A），以使这种SpCas 9核酸酶在lentiCRISPR v2载体中死亡，命名为dCas 9。然后，将靶向人MDM 2基因的第二启动子的MDM 2-sgRNA克隆到该载体中，用于产生慢病毒以感染携带MDM 2（SNP 309）（T/G）的杂合基因型的人视网膜色素上皮（RPE）细胞。lacZ-sgRNA用作对照。结果，我们发现，与具有dCas 9/MDM 2-sgRNA的RPE细胞相比，来自实验兔的玻璃体在具有dCas 9/lacZ-sgRNA的RPE细胞中诱导Mdm 2增加1.9 +/-0.2倍和p53减少2.0 +/-0.2倍，这表明在MDM 2-sgRNA指导下的dCas 9阻止了RV刺激的Mdm 2增加。此外，我们发现，与使用dCas 9/MDM 2-sgRNA的细胞相比，兔玻璃体显著增强了使用dCas 9/lacZ-sgRNA的细胞的细胞增殖（1.5 +/-0.2倍）、抗凋亡存活（2.2 +/- 0.2倍）、迁移（10 +/- 1.5%）和收缩（112.7 +/- 14.1 mm（2））。这些结果表明，靶向MDM 2的P2的dCas 9的应用是由于MDM 2的P2驱动的异常表达引起的疾病（例如增殖性玻璃体视网膜病变）的潜在治疗方法。
Mouse double minute (MDM)2 single nucleotide polymorphism (SNP) 309G allele in the second promoter of MDM2 enhances vitreous-induced expression of Mdm2 and degradation of the tumor suppressor protein p53. This MDM2(SNP309G) contributes to certain cancer development and experimental proliferative vitreoretinopathy. The goal of this study is to discover a novel strategy to only block vitreous-induced expression of Mdm2 for preventing vitreous-induced cell proliferation and survival and thus find a potential novel strategy to treat proliferation-related diseases. We created two mutations (D10A and H840A) in Streptococcus pyogenes (Sp)Cas9 within the nuclease domains (RuvC1 and HNH, respectively) to render this SpCas9 nuclease dead named as dCas9 in a lentiCRISPR v2 vector. Then an MDM2-sgRNA targeting the second promoter of human MDM2 gene was cloned into this vector for producing lentivirus to infect human retinal pigment epithelial (RPE) cells with, which carry a heterozygous genotype of MDM2(SNP309) (T/G). lacZ-sgRNA was used as a control. As a result, we discovered that vitreous from experimental rabbits induced a 1.9 +/- 0.2 fold increase in Mdm2 and a 2.0 +/- 0.2 fold decrease in p53 in the RPE cells with dCas9/lacZ-sgRNA compared to those with dCas9/MDM2-sgRNA, suggesting that dCas9 under the guidance of the MDM2-sgRNA prevented RV-stimulated increase in Mdm2. In addition, we found that the rabbit vitreous significantly enhanced cell proliferation (1.5 +/- 0.2 fold), survival against apoptosis (2.2 +/- 0.2 fold), migration (10 +/- 1.5%) and contraction (112.7 +/- 14.1 mm(2)) of the cells with dCas9/lacZ-sgRNA compared with those with dCas9/MDM2-sgRNA. These results indicated that application of the dCas9 targeted to the P2 of MDM2 is a potential therapeutic approach to diseases due to the P2-driven aberrant expression of Mdm2 - such as proliferative vitreoretinopathy.