METTL3的抑制通过恢复奈普赖氨酸的表达来减轻LPS诱导的肺泡上皮细胞凋亡和急性肺损伤。

IF 5.2 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL
Jingsi Jia , Yu Yuan , Yi He , Binaya Wasti , Wentao Duan , Zhifeng Chen , Danhong Li , Wenjin Sun , Qingping Zeng , Libing Ma , Xiufeng Zhang , Shaokun Liu , Dongshan Zhang , Linxia Liu , Qimi Liu , Hengxing Liang , Guyi Wang , Xudong Xiang , Bing Xiao
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LPS treatment on mouse lung epithelial 12 (MLE-12) cell was applied to establish an in vitro model of LPS-induced ALI. H&amp;E staining, lung wet-to-dry ratio, and total broncho-alveolar lavage fluid (BALF) concentrations were used to evaluate lung injury. Overall, the m6A level was determined with the m6A RNA Methylation Quantification Kit and dot blot assay. Expression of METTL3 and neprilysin were measured with immunohistochemistry, immunofluorescence, immunofluorescence-fluorescence in situ hybridization, and western blot. Apoptosis was detected with TUNEL, western blot, and flow cytometry. The interaction of METTL3 and neprilysin was determined with RIP-qPCR and MeRIP.</p></div><div><h3>Key findings</h3><p>METTL3 expression and apoptosis were increased in alveolar epithelial cells of mice treated with LPS, and METTL3-CKO or METTL3 inhibitor STM2457 could alleviate apoptosis and LPS-induced ALI. In MLE-12 cells, LPS-Induced METTL3 expression and apoptosis. 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引用次数: 0

摘要

目的:探讨甲基转移酶样3(METTL3)在脂多糖(LPS)诱导的急性肺损伤(ALI)发病机制中的作用。此外,还使用了METTL3抑制剂STM2457。应用LPS处理小鼠肺上皮12(MLE-12)细胞,建立LPS诱导的ALI体外模型。H&E染色、肺湿干比和支气管肺泡灌洗液(BALF)总浓度用于评估肺损伤。总体而言,使用m6A RNA甲基化定量试剂盒和斑点印迹测定法测定m6A水平。采用免疫组织化学、免疫荧光、免疫荧光原位杂交和蛋白质印迹法检测METTL3和奈普赖氨酸的表达。用TUNEL、蛋白质印迹和流式细胞术检测细胞凋亡。用RIP-qPCR和MeRIP检测了METTL3和奈普赖氨酸的相互作用。关键发现:经LPS处理的小鼠肺泡上皮细胞中METTL3的表达和凋亡增加,METTL3-CKO或METTL3抑制剂STM2457可减轻细胞凋亡和LPS诱导的ALI。在MLE-12细胞中,LPS诱导METTL3的表达和细胞凋亡。METTL3的敲除减轻,而METTL3的过表达加剧了LPS诱导的细胞凋亡。LPS处理降低了奈普赖氨酸的表达,奈普赖素表达的干预在不影响METTL3表达的情况下负调控细胞凋亡,并减轻了METTL3对LPS诱导的细胞凋亡的促进作用。此外,METTL3可以与奈普赖氨酸的mRNA结合,并降低其表达。意义:我们的研究结果表明,抑制METTL3可以通过恢复奈普赖氨酸的表达来发挥抗细胞凋亡和ALI保护作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Inhibition of METTL3 alleviated LPS-induced alveolar epithelial cell apoptosis and acute lung injury via restoring neprilysin expression

Aims

To investigate the role and mechanisms of methyltransferase-like 3 (METTL3) in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury (ALI).

Main methods

LPS intratracheally instillation was applied in alveolar epithelial cell METTL3 conditional knockout (METTL3-CKO) mice and their wild-type littermates. In addition, METTL3 inhibitor STM2457 was used. LPS treatment on mouse lung epithelial 12 (MLE-12) cell was applied to establish an in vitro model of LPS-induced ALI. H&E staining, lung wet-to-dry ratio, and total broncho-alveolar lavage fluid (BALF) concentrations were used to evaluate lung injury. Overall, the m6A level was determined with the m6A RNA Methylation Quantification Kit and dot blot assay. Expression of METTL3 and neprilysin were measured with immunohistochemistry, immunofluorescence, immunofluorescence-fluorescence in situ hybridization, and western blot. Apoptosis was detected with TUNEL, western blot, and flow cytometry. The interaction of METTL3 and neprilysin was determined with RIP-qPCR and MeRIP.

Key findings

METTL3 expression and apoptosis were increased in alveolar epithelial cells of mice treated with LPS, and METTL3-CKO or METTL3 inhibitor STM2457 could alleviate apoptosis and LPS-induced ALI. In MLE-12 cells, LPS-Induced METTL3 expression and apoptosis. Knockdown of METTL3 alleviated, while overexpression of METTL3 exacerbated LPS-induced apoptosis. LPS treatment reduced neprilysin expression, the intervention of neprilysin expression negatively regulated apoptosis without affecting METTL3 expression, and mitigated the promoting effect of METTL3 on LPS-induced apoptosis. Additionally, METTL3 could bind to the mRNA of neprilysin, and reduce its expression.

Significance

Our findings revealed that inhibition of METTL3 could exert anti-apoptosis and ALI-protective effects via restoring neprilysin expression.

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来源期刊
Life sciences
Life sciences 医学-药学
CiteScore
12.20
自引率
1.60%
发文量
841
审稿时长
6 months
期刊介绍: Life Sciences is an international journal publishing articles that emphasize the molecular, cellular, and functional basis of therapy. The journal emphasizes the understanding of mechanism that is relevant to all aspects of human disease and translation to patients. All articles are rigorously reviewed. The Journal favors publication of full-length papers where modern scientific technologies are used to explain molecular, cellular and physiological mechanisms. Articles that merely report observations are rarely accepted. Recommendations from the Declaration of Helsinki or NIH guidelines for care and use of laboratory animals must be adhered to. Articles should be written at a level accessible to readers who are non-specialists in the topic of the article themselves, but who are interested in the research. The Journal welcomes reviews on topics of wide interest to investigators in the life sciences. We particularly encourage submission of brief, focused reviews containing high-quality artwork and require the use of mechanistic summary diagrams.
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