Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

Deqiang Luo,Xiaojin Feng,Wei Dai,Fen Liu,Kejian Qian,Ning Zhao,Rui Xiao,Qiang Shao,Ying Yang,Wei Peng,Yamei Cui,Chengzhi Ding

doi:10.1038/s41419-021-04180-y

Abstract

Acute lung injury (ALI) is a common lung pathology that is accompanied by alveolar macrophage (AM) activation and inflammatory response. This study investigated the role of the long non-coding RNA NONRATT004344 (hereafter named lncRNA NLRP3) in regulating the Nod-like receptor protein 3 (NLRP3)-triggered inflammatory response in early ALI and the underlying mechanism as well. We established LPS-induced ALI models to explore their interactive mechanisms in vitro and in vivo. Luciferase reporter assays were performed to determine that miR-138-5p could bind to lncRNA NLRP3 and NLRP3. We observed increased lncRNA NLRP3 expression, decreased miR-138-5p expression, NLRP3 inflammasome activation, and upregulated caspase-1, IL-1β, and IL-18 expression in the LPS-induced ALI model. Furthermore, lncRNA NLRP3 overexpression activated the NLRP3 inflammasome and promoted IL-1β and IL-18 secretion; the miR-138-5p mimic abolished these effects in vivo and in vitro. Consistently, miR-138-5p inhibition reversed the effects of lncRNA NLRP3 silencing on the expression of NLRP3-related molecules and inhibition of the NLRP3/caspase-1/IL-1β signalling pathway. Mechanistically, lncRNA NLRP3 sponging miR-138-5p facilitated NLRP3 activation through a competitive endogenous RNA (ceRNA) mechanism. In summary, our results suggested that lncRNA NLRP3 binding miR-138-5p promotes NLRP3-triggered inflammatory response via lncRNA NLRP3/miR-138-5p/NLRP3 ceRNA network (ceRNET) and provides insights into the treatment of early ALI.

Highlights

Acute lung injury (ALI)/mild acute respiratory distress syndrome (ARDS) in Berlin definition, which is accompanied by alveolar macrophage (AM) activation and inflammatory response, often progresses to an inflammatory storm that develops into severe ARDS and patient death [1, 2]
Our study found that lncRNA Nod-like receptor protein 3 (NLRP3) sponges miR-138-5p to facilitate the NLRP3triggered inflammatory response via the lncRNA NLRP3/miR-1385p/NLRP3 ceRNA network (ceRNET), and lncRNA NLRP3 and miR-138-5p provide before protein extraction (n = 6)
The quantitative reverse-transcriptase PCR (qRT-PCR) results showed that the protein expression levels of NLRP3 and caspase-1 confirmed expression of NLRP3, caspase-1, IL-18, and IL-1β was significantly the qRT-PCR results (P < 0.05, Fig. 2K). These results showed that lncRNA NLRP3, NLRP3, caspase-1, IL-1β, and IL-18 expression were most significantly increased after 6 h of LPS treatment and miR138-5p expression was most significantly decreased at this upregulated by miR-138-5p inhibition, and miR-138-5p mimics inhibited the mRNA expression of these genes (P < 0.05, Fig. 5C–G)

Summary

INTRODUCTION

Acute lung injury (ALI)/mild acute respiratory distress syndrome (ARDS) in Berlin definition, which is accompanied by alveolar macrophage (AM) activation and inflammatory response, often progresses to an inflammatory storm that develops into severe ARDS and patient death [1, 2]. We predicted that with RNA sequencing (RNA-seq) and bioinformatics analysis, the lncRNA NONRATT004344/miR-1385p/NLRP3 ceRNA network (ceRNET) could regulate NLRP3triggered inflammatory responses in ALI [31]. The extracted RNA expression levels of lncRNA NLRP3 and β-actin (nuclear control transcript and cytoplasmic control transcript) from each fraction were analysed by qRT-PCR. Histopathological analysis Haematoxylin and eosin (HE) staining was used to analyse the Western blotting Total protein extraction from NR8383 AM cells (5 × 106 cells) and rat lungs were performed with radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher Scientific, Shanghai City, China). The primary antibodies used in this experiment included was used to measure albumin levels (EK0592 and Solarbio Life Science; rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti- SEKR-0009; ELISA; Boster Biotech). The binding sites of miR-138-5p in the NLRP3 gene promoter were identified using the same method mentioned above

RESULTS

Luo et al 4

DISCUSSION

ETHICS APPROVAL AND CONSENT TO PARTICIPATE