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• W2954552022 endingPage "107716" @default.
• W2954552022 startingPage "107716" @default.
• W2954552022 abstract "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 MDM2SNP309G 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 MDM2SNP309 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 mm2) 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." @default.
• W2954552022 created "2019-07-12" @default.
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• W2954552022 date "2019-09-01" @default.
• W2954552022 modified "2023-09-25" @default.
• W2954552022 title "Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells" @default.
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• W2954552022 doi "https://doi.org/10.1016/j.exer.2019.107716" @default.
• W2954552022 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31278903" @default.
• W2954552022 hasPublicationYear "2019" @default.
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