牛颗粒细胞体外接触四氢大麻酚会影响 DNA 甲基化,但不会影响 microRNA 的表达。

IF 2.8 3区 医学 Q2 PHARMACOLOGY & PHARMACY
Sabrina Floccari, Reem Sabry, Laurie Choux, Michael S Neal, Jibran Y Khokhar, Laura A Favetta
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引用次数: 0

摘要

背景:全球大麻使用量的增加引发了大麻对生育的影响问题。育龄妇女中大麻消费量的增加日益引起人们的关注,因为这一群体在生殖健康方面非常脆弱。大量证据表明,大麻的精神活性成分Δ9-四氢大麻酚(THC)与内源性大麻素系统(ECS)相互作用,有助于调节哺乳动物的生殖。本研究旨在通过以下方法研究 THC 在牛颗粒细胞(GCs)中的表观遗传效应:(1)通过测量 5-mC 和 5-hmC 水平研究 DNA 的整体甲基化;(2) 测量关键的甲基化调节因子,包括甲基化酶 DNMT1、DNMT3a、DNMT3b 以及去甲基化酶 TDG 和 TET1/2/3;以及 (3) 评估发育能力中与生育力相关的关键 miRNA,包括 miR-21、-155、-33b、-324 和 -346。方法:牛 GCs 被用作人类生殖毒性的转化模型。为确定四氢大麻酚的影响,从牛卵巢的积聚-卵母细胞-复合体(COCs)中分离出 GCs,体外培养 7 天或直到汇合,并在 1 胞期(P1)低温保存。在实验中,解冻细胞,培养至第 2 期(P2),限制血清 24 小时,并在以下五组中选择一组处理 24 小时:对照组、载体组(1:1:18 乙醇:吐温:生理盐水)和三种临床相关 THC 剂量组(0.032、0.32 和 3.2 μM)。通过流式细胞术测量 5-mC 和 5-hmC 的水平来评估全局甲基化。为了评估甲基化调节因子和 miRNA 的 mRNA 和蛋白质表达情况,采用了 qPCR 和 Western 印迹技术。采用 Shapiro-Wilk 检验确定数据集的正态性。使用 GraphPad Prism 6.0.0 进行单因素方差分析,以确定统计显著性:结果表明,暴露于低浓度 THC 后,5-mC 水平明显下降(p = 0.0435),而 5-hmC 水平没有变化。暴露于高浓度 THC 后,DNMT1 在 RNA 水平上明显升高(p 结论:该研究表明,THC 对人体的影响主要集中在细胞内:这项研究表明,四氢大麻酚主要影响 DNA 甲基化,但不影响 miRNA 图谱,最终改变基因表达,并可能损害卵母细胞的能力、成熟度和受精潜能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
DNA methylation, but not microRNA expression, is affected by in vitro THC exposure in bovine granulosa cells.

Background: A global increase in cannabis use has led to questions about its effects on fertility. The rise in consumption amongst women of reproductive age is a growing concern, as this group is vulnerable in terms of reproductive health. Ample evidence suggests that the psychoactive component of cannabis, Δ9-Tetrahydrocannabinol (THC), interacts with the endocannabinoid system (ECS), that helps regulate mammalian reproduction. This study aimed to research the epigenetic effects of THC in bovine granulosa cells (GCs) by (1) investigating global DNA methylation via measuring 5-mC and 5-hmC levels; (2) measuring key methylation regulators, including the methylating enzymes DNMT1, DNMT3a, DNMT3b and the demethylases TDG and TET1/2/3; and (3) assessing fertility-associated miRNAs key in developmental competency, including miR-21, -155, -33b, -324 and -346.

Methods: Bovine GCs were used as a translational model for reproductive toxicity in humans. To determine THC effects, GCs were isolated from Cumulus-Oocyte-Complexes (COCs) from bovine ovaries, cultured in vitro for 7 days, or until confluent, and cryopreserved at passage 1 (P1). For experimentation, cells were thawed, cultured until passage 2 (P2), serum restricted for 24-h and treated for 24-h in one of five groups: control, vehicle (1:1:18 ethanol: tween: saline) and three clinically relevant THC doses (0.032, 0.32 and 3.2 μM). Global methylation was assessed by measuring 5-mC and 5-hmC levels with flow cytometry. To assess mRNA and protein expression of methylation regulators and miRNA profiles, qPCR and Western Blotting were utilized. Shapiro-Wilk test was used to determine normality within datasets. One-way ANOVA was applied to determine statistical significance using GraphPad Prism 6.0.0.

Results: Results indicate a significant decrease (p = 0.0435) in 5-mC levels following low THC exposure, while no changes were observed in 5-hmC levels. A significant increase in DNMT1 following high THC exposure at the RNA level (p < 0.05) and a significant increase following low THC exposure at the protein level (p = 0.0048) were also observed. No significant differences were observed in DNMT3a/3b, TDG, TET1/2/3 mRNAs or in any of the miRNAs analyzed.

Conclusions: This research suggests that THC mainly affects DNA methylation, but not miRNA profiles, ultimately altering gene expression and likely impairing oocyte competence, maturation, and fertilization potential.

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来源期刊
BMC Pharmacology & Toxicology
BMC Pharmacology & Toxicology PHARMACOLOGY & PHARMACYTOXICOLOGY&nb-TOXICOLOGY
CiteScore
4.80
自引率
0.00%
发文量
87
审稿时长
12 weeks
期刊介绍: BMC Pharmacology and Toxicology is an open access, peer-reviewed journal that considers articles on all aspects of chemically defined therapeutic and toxic agents. The journal welcomes submissions from all fields of experimental and clinical pharmacology including clinical trials and toxicology.
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