ATP2B1-AS1 通过调控 miR-23a-3p/TLR4 轴加剧脓毒症诱导的细胞凋亡和炎症反应。

ATP2B1-AS1 exacerbates sepsis-induced cell apoptosis and inflammation by regulating miR-23a-3p/TLR4 axis.

机构信息

Department of Pediatrics, the First Affiliated Hospital of Chengdu Medical College, Chengdu, Sichuan, China;

Department of Pediatrics, the First Affiliated Hospital of Chengdu Medical College, Chengdu, Sichuan, China.

出版信息

Allergol Immunopathol (Madr). 2023 Mar 1;51(2):17-26. doi: 10.15586/aei.v51i2.782. eCollection 2023.

DOI:10.15586/aei.v51i2.782

PMID:36916084

Abstract

BACKGROUND

Sepsis is a life-threatening disease with dominant mortality. Its early diagnosis and treatment can improve prognosis and reduce mortality. Long noncoding RNAs (lncRNAs) ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1-AS1) is dysregulated and is involved in the progression of various diseases. Nevertheless, the role of ATP2B1-AS1 in sepsis remains unclear.

METHODS

A human monocytic cell line, THP-1 cells, was stimulated to induce a model of sepsis . The levels of ATP2B1-AS1, miR-23a-3p, and TLR4 were assessed by real-time quantitative polymerase chain reaction. The role of ATP2B1-AS1 in cell apoptosis and inflammation was explored by flow cytometry, Western blot analysis and enzyme-linked immunosorbent serologic assay. The binding sites between ATP2B1-AS1 and miR-23a-3p, and between miR-23a-3p and TLR4 were predicted by BiBiServ and the Encyclopedia of RNA Interactomes (ENCORI) online sites, respectively, and confirmed by the luciferase assay.

RESULTS

The level of ATP2B1-AS1 was increased in lipopolysaccharide (LPS)-treated THP-1 cells. LPS increased apoptosis ratio, relative protein expressions of pro-apoptotic factors, and relative messenger RNA (mRNA) level and concentrations of pro-inflammatory cytokines, but decreased the relative expression of anti-apoptosis protein and relative mRNA level and concentrations of anti-inflammatory factor. All these alterations were reversed with transfection of shATP2B1-AS1 into THP-1 cells. Moreover, ATP2B1-AS1 directly bound miR-23a-3p and negatively modulated the level of miR-23a-3p. Meanwhile, TLR4 was directly targeted by miR-23a-3p, and negatively and positively modulated by miR-23a-3p and ATP2B1-AS1, respectively.

CONCLUSION

ATP2B1-AS1 aggravated apoptosis and inflammation by modulating miR-23a-3p/TLR4 axis in LPS-treated THP-1 cells.

摘要

背景

脓毒症是一种具有高死亡率的危及生命的疾病。早期诊断和治疗可以改善预后，降低死亡率。长链非编码 RNA（lncRNA）ATP 酶质膜 Ca2+转运 1 反义 RNA 1（ATP2B1-AS1）失调，并参与各种疾病的进展。然而，ATP2B1-AS1 在脓毒症中的作用尚不清楚。

方法

用脂多糖（LPS）刺激人单核细胞系 THP-1 细胞，诱导脓毒症模型。实时定量聚合酶链反应检测 ATP2B1-AS1、miR-23a-3p 和 TLR4 的水平。通过流式细胞术、Western blot 分析和酶联免疫吸附试验探讨 ATP2B1-AS1 对细胞凋亡和炎症的作用。通过 BiBiServ 和 Encyclopedia of RNA Interactomes（ENCORI）在线网站分别预测 ATP2B1-AS1 与 miR-23a-3p 以及 miR-23a-3p 与 TLR4 的结合位点，并通过荧光素酶报告基因实验进行验证。

结果

LPS 处理的 THP-1 细胞中 ATP2B1-AS1 水平升高。LPS 增加了凋亡比例、促凋亡因子的相对蛋白表达以及促炎细胞因子的相对信使 RNA（mRNA）水平和浓度，但降低了抗凋亡蛋白的相对表达和抗炎因子的相对 mRNA 水平和浓度。所有这些改变都可以通过转染 shATP2B1-AS1 到 THP-1 细胞中得到逆转。此外，ATP2B1-AS1 直接与 miR-23a-3p 结合，并负调控 miR-23a-3p 的水平。同时，TLR4 被 miR-23a-3p 直接靶向，并被 miR-23a-3p 以及 ATP2B1-AS1 分别负向和正向调控。