背景:肿瘤中钙电穿孔(CaEP)后Ca2+稳态的破坏已被证明可引起增强的抗肿瘤作用,并对健康组织产生不同的影响,如内皮。因此,我们的研究旨在确定在Ca2+动力学和基因表达参与调节Ca2+信号和稳态的差异,以及CaEP对已建立的内皮细胞系EA的细胞骨架和粘附连接的影响。hy926和HMEC-1。
方法:对EA进行CaEP。hy926和HMEC-1细胞随着Ca2+浓度的增加。使用PrestoBlue评估CaEP后的生存力,而对细胞骨架和粘附连接的影响是通过免疫荧光染色(F-肌动蛋白,α-微管蛋白,VE-钙粘蛋白)。细胞内Ca2调节([Ca2]i)的差异通过使用Fura-2-AM的荧光光谱测量来确定,将细胞暴露于DPBS,离子霉素,thapsigargin,ATP,缓激肽,血管紧张素II,乙酰胆碱,LaCl3和GdCl3。通过RNA测序分析与细胞骨架和Ca2信号传导相关的差异表达基因和途径来鉴定分子差异。
结果:EA。hy926细胞,在增加Ca2+浓度时,与HMEC-1相比,显示更高的CaEP易感性和更低的生存率。免疫荧光证实CaEP诱导,EA的时间和Ca2+依赖性形态变化。hy926的肌动蛋白丝,微管,和细胞-细胞连接。荧光光谱法Ca2动力学在EA中显示出更高的Ca2响应幅度。hy926暴露于缓冲液,G蛋白偶联受体激动剂,缓激肽,和血管紧张素II与HMEC-1相比。离子霉素暴露后,HMEC-1表现出明显更高的[Ca2]i变化,而对thapsigargin的回应,ATP,和乙酰胆碱在两种细胞系中相似。没有细胞外Ca2离子的ATP在EA中诱导了显着更高的[Ca2]i升高。hy926,提示嘌呤能离子型P2X和代谢型P2Y受体激活。RNA测序分析显示细胞骨架和Ca2+相关基因表达存在显著差异,强调EA中ORAI2,TRPC1,TRPM2,CNGA3,TRPM6的上调以及TRPV4和TRPC4的下调。hy926与HMEC-1.此外,KEGG分析显示EA中Ca2输入上调,输出基因下调。hy926.
结论:我们的发现表明,EA之间在CaEP反应和[Ca2+]i调节方面存在显著差异。hy926和HMEC-1,这可能归因于不同的转录组概况。EA.hy926与HMEC-1相比,对[Ca2+]i变化具有更高的敏感性和敏感性,这可能与Ca2相关基因的过表达有关,并且无法减轻[Ca2]i的变化。该研究为根据研究目标选择EC模型提供了生物信息学基础。
Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1.
CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing.
EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926\'s actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926.
Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.