Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508, USA.
Institute for Digestive System Research, Lithuanian University of Health Sciences, 44307 Kaunas, Lithuania.
Int J Mol Sci. 2023 May 24;24(11):9191. doi: 10.3390/ijms24119191.
The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in YP uptake. The respective proteins could be part of electropermeabilization lesions or increase their lifespan. In contrast, as many as 39 genes were identified as likely hits for the increased YP uptake, meaning that the respective proteins contributed to membrane stability or repair after nsEP. The expression level of eight genes in different types of human cells showed strong correlation (R > 0.9, < 0.02) with their LD for lethal nsEP treatments, and could potentially be used as a criterion for the selectivity and efficiency of hyperplasia ablations with nsEP.
本研究旨在鉴定内源性蛋白，这些蛋白在细胞膜被 nsEP（20 或 40 个脉冲，300ns 宽度，7kV/cm）破坏时协助或阻碍其通透性状态。我们采用 LentiArray CRISPR 文库生成了稳定表达 Cas9 核酸酶的人单核细胞系 U937 中 316 个编码膜蛋白的基因的敲除（KO）。通过 Yo-Pro-1（YP）染料摄取来测量 nsEP 引起的细胞膜通透性增加，并与假暴露 KO 和转导非靶向（乱序）gRNA 的对照细胞进行比较。只有 SCNN1A 和 CLCA1 基因的两个 KO 显示出 YP 摄取的统计学显著减少。相应的蛋白质可能是电穿孔损伤的一部分或延长其寿命。相比之下，多达 39 个基因被鉴定为 YP 摄取增加的可能命中，这意味着相应的蛋白质有助于 nsEP 后的膜稳定性或修复。八种不同类型人类细胞的基因表达水平与它们对致死性 nsEP 处理的 LD 之间存在强烈相关性（R>0.9， <0.02），并且可能可作为 nsEP 治疗细胞过度增殖的选择性和效率的标准。
