NUTM2A-AS1 沉默通过靶向 let-7c-5p/HMGB1 轴缓解脂多糖诱导的牙髓细胞凋亡和炎症。

NUTM2A-AS1 silencing alleviates LPS-induced apoptosis and inflammation in dental pulp cells through targeting let-7c-5p/HMGB1 axis.

机构信息

Department of Oral & Cranio-Maxillofacial Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai 200011, China.

出版信息

Int Immunopharmacol. 2021 Jul;96:107497. doi: 10.1016/j.intimp.2021.107497. Epub 2021 Apr 5.

DOI:10.1016/j.intimp.2021.107497

PMID:33831808

Abstract

BACKGROUND

Long non-coding RNA (lncRNA) NUTM2A antisense RNA 1 (NUTM2A-AS1) has been reported to be abnormally up-regulated in pulpitis tissues. However, the function of NUTM2A-AS1 in pulpitis remains unclear. The aim of this study was to investigate the role and working mechanism of NUTM2A-AS1 in pulpitis using lipopolysaccharide (LPS)-treated human dental pulp cells (HDPCs).

METHODS

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and lactate dehydrogenase (LDH) release detection assay were conducted to analyze the viability of HDPCs. Cell inflammatory response was analyzed through measuring the protein levels of interleukin-6 (IL-6) and IL-8. Western blot assay and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to measure protein expression and RNA expression, respectively. Bioinformatic database StarBase was used to predict the possible targets of NUTM2A-AS1 and let-7c-5p, and dual-luciferase reporter assay was conducted to verify these intermolecular interactions.

RESULTS

LPS stimulation restrained cell viability and induced cell apoptosis and inflammation of HDPCs. LPS exposure up-regulated the expression of NUTM2A-AS1 and High-Mobility Group Box 1 (HMGB1) and down-regulated the level of let-7c-5p. LPS-induced injury in HDPCs was partly attenuated by the silencing of NUTM2A-AS1 or HMGB1. Let-7c-5p was confirmed as a direct target of NUTM2A-AS1, and let-7c-5p bound to the 3' untranslated region (3'UTR) of HMGB1 messenger RNA (mRNA) in HDPCs. HMGB1 overexpression largely overturned NUTM2A-AS1 silencing-mediated effects in LPS-induced HDPCs.

CONCLUSION

NUTM2A-AS1 knockdown attenuated LPS-induced damage in HDPCs partly through targeting let-7c-5p/HMGB1 axis.

摘要

背景

长链非编码 RNA（lncRNA）NUTM2A 反义 RNA 1（NUTM2A-AS1）在牙髓组织中被报道异常上调。然而，NUTM2A-AS1 在牙髓炎中的作用尚不清楚。本研究旨在通过脂多糖（LPS）处理的人牙髓细胞（HDPC）来研究 NUTM2A-AS1 在牙髓炎中的作用和作用机制。

方法

采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法、流式细胞术和乳酸脱氢酶（LDH）释放检测法分析 HDPC 的活力。通过测量白细胞介素-6（IL-6）和 IL-8 的蛋白水平分析细胞炎症反应。采用 Western blot 法和实时定量聚合酶链反应（qRT-PCR）分别测定蛋白表达和 RNA 表达。生物信息学数据库 StarBase 用于预测 NUTM2A-AS1 和 let-7c-5p 的可能靶点，并通过双荧光素酶报告基因检测验证这些分子间相互作用。

结果

LPS 刺激抑制细胞活力并诱导 HDPC 细胞凋亡和炎症。LPS 暴露上调 NUTM2A-AS1 和高迁移率族蛋白 B1（HMGB1）的表达，下调 let-7c-5p 的水平。NUTM2A-AS1 或 HMGB1 的沉默部分减轻了 LPS 诱导的 HDPC 损伤。let-7c-5p 被证实是 NUTM2A-AS1 的直接靶点，并且 let-7c-5p 在 HDPC 中与 HMGB1 信使 RNA（mRNA）的 3'非翻译区（3'UTR）结合。HMGB1 的过表达在很大程度上推翻了 LPS 诱导的 HDPC 中 NUTM2A-AS1 沉默介导的作用。