n6 -甲基腺苷RNA碱基修饰调节nkg2d依赖性和细胞毒性基因在自然杀伤细胞中的表达。

IF 2.1 4区 医学 Q3 GENETICS & HEREDITY
Raghda A Elsabbagh, Ghada Abdelhady, Doris Urlaub, Mina Sandusky, Ola Khorshid, Mohamed Z Gad, Khaled Abou-Aisha, Carsten Watzl, Mona Rady
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引用次数: 0

摘要

背景:乳腺癌(BC)是女性中最常见的癌症。n6 -甲基腺苷(m6A)是哺乳动物mrna中最常见的内部修饰,在各种生物过程中起着至关重要的作用。然而,其在BC自然杀伤细胞(NK)中的功能尚不清楚。NK细胞对癌症免疫监测至关重要。本研究旨在评估BC患者NK细胞中NKG2D细胞毒性信号通路相关转录本中的m6A水平,并找出其对mRNA水平的影响。此外,它还评估了故意改变NK细胞中m6A水平如何影响NKG2D途径基因的mRNA和蛋白质表达以及NK细胞功能。方法:采用甲基化RNA免疫沉淀-逆转录pcr (MERIP-RT-PCR)检测BC患者和对照组nkg2d通路相关基因转录本中的m6A甲基化。为了有意改变原代培养的人NK细胞中的m6A水平,使用CRISPR-CAS9系统敲除m6A去甲基化酶FTO和ALKBH5,并使用甲氯芬酸(MA)抑制FTO。分别采用RT-qPCR、Western blot或流式细胞术评估m6A改变对相应mRNA和蛋白水平的影响。此外,通过脱颗粒和51Cr释放细胞毒性试验评估NK细胞功能。结果:与对照组相比,NKG2D转录本(一种检测应激非自身肿瘤细胞的激活受体)在BC患者的3‘非翻译区(3’ utr)中具有显着更高的m6A水平,同时相应的mRNA水平显着降低。相反,与对照组相比,BC患者中ERK2和PRF1转录本的m6A水平显著降低,mRNA表达水平较高。在BC患者中,PI3K、PAK1和GZMH mRNA水平也显著升高。此外,通过MA在培养的原代NK细胞中人为增加转录本的m6A水平,降低了NKG2D途径基因和死亡受体配体的mRNA水平,但不影响蛋白质表达或NK细胞功能。结论:3'UTR区m6A水平较高的转录本数量较少,反之亦然。然而,靶基因mRNA水平的变化并不影响其相应的蛋白水平或NK细胞功能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
N6-methyladenosine RNA base modification regulates NKG2D-dependent and cytotoxic genes expression in natural killer cells.

Background: Breast cancer (BC) is the most commonly diagnosed cancer in women. N6-methyladenosine (m6A) is the most prevalent internal modification in mammalian mRNAs and plays a crucial role in various biological processes. However, its function in Natural killer (NK) cells in BC remains unclear. NK cells are essential for cancer immunosurveillance. This study aims to assess m6A levels in transcripts involved in the NKG2D cytotoxicity signaling pathway in NK cells of BC patients compared to controls and find out its impact on mRNA levels. Additionally, it evaluates how deliberately altering m6A levels in NK cells affects mRNA and protein expression of NKG2D pathway genes and NK cell functionality.

Methods: m6A methylation in transcripts of NKG2D-pathway-related genes in BC patients and controls was determined using methylated RNA immunoprecipitation-reverse transcription-PCR (MERIP-RT-PCR). To deliberately alter m6A levels in primary cultured human NK cells, the m6A demethylases, FTO and ALKBH5, were knocked out using the CRISPR-CAS9 system, and FTO was inhibited using Meclofenamic acid (MA). The impact of m6A alteration on corresponding mRNA and protein levels was assessed using RT-qPCR and Western blot analysis or flow cytometry, respectively. Additionally, NK cell functionality was evaluated through degranulation and 51Cr release cytotoxicity assays.

Results: Transcripts of NKG2D, an activating receptor that detects stressed non-self tumour cells, had significantly higher m6A levels in the 3' untranslated region (3'UTR) accompanied by a marked reduction in their corresponding mRNA levels in BC patients compared to controls. Conversely, transcripts of ERK2 and PRF1 exhibited significantly lower m6A levels escorted with higher mRNA expression in BC patients relative to controls. The mRNA levels of PI3K, PAK1 and GZMH were also significantly elevated in BC patients. Furthermore, artificially increasing transcripts' m6A levels via MA in cultured primary NK cells reduced mRNA levels of NKG2D pathway genes and death receptor ligands but did not affect protein expression or NK cell functionality.

Conclusion: Transcripts with higher m6A levels in the 3'UTR region were less abundant, and vice versa. However, changes in mRNA levels of the target genes didn't impact their corresponding protein levels or NK cell functionality.

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来源期刊
BMC Medical Genomics
BMC Medical Genomics 医学-遗传学
CiteScore
3.90
自引率
0.00%
发文量
243
审稿时长
3.5 months
期刊介绍: BMC Medical Genomics is an open access journal publishing original peer-reviewed research articles in all aspects of functional genomics, genome structure, genome-scale population genetics, epigenomics, proteomics, systems analysis, and pharmacogenomics in relation to human health and disease.
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