Silencing of lncRNA Gm26917 Attenuates Alveolar Macrophage-mediated Inflammatory Response in LPS-induced Acute Lung Injury Via Inhibiting NKRF Ubiquitination

Abstract

The inflammatory response mediated by alveolar macrophages plays a crucial role in the development of acute lung injury. Numerous studies have reported that lncRNAs are highly expressed in acute lung injury in mouse models and cell lines, and acute lung injury (ALI) can be effectively alleviated by targeting these lncRNAs. The aim of this study was to explore the mechanism by LncRNA Gm26917 regulates the inflammatory response in alveolar macrophages during acute lung injury mouse model. We initially observed a significant upregulation of Gm26917 expression in both ALI conditions and in MH-S cells treated with LPS. Furthermore, the silencing of Gm26917 via lentivirus-mediated methods conferred protection against LPS-induced ALI. Additionally, siRNA-mediated knockdown of Gm26917 attenuated LPS-induced inflammatory responses and modulated the function of alveolar macrophages. Subsequent mechanistic studies revealed that Gm26917 interacts with NKRF, and its knockdown suppressed NKRF ubiquitination, thereby enhancing NKRF binding to p50 and subsequently inhibiting the NF-κB signaling pathway. In conclusion, our findings demonstrate that silencing Gm26917 can mitigate LPS-induced ALI by modulating the NF-κB signaling pathway in alveolar macrophages through interactions with NKRF.

Introduction

There are a variety of intrapulmonary and extrapulmonary factors that may contribute to acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) [1]. Among these factors, sepsis is the most common cause of death, and sepsis-induced acute lung injury can progress to ARDS in later stages, with a mortality rate of up to 40% [2]. Alveolar macrophages (AMs), the most common immune cells in the alveoli, play an essential role in regulating lung homeostasis [3]. When the lung is exposed to external stimuli, alveolar macrophages serve as the first line of immune defense against inflammation through phagocytosis, removal of endogenous proteins and cellular debris [4]. Moreover, in a lung injury model, due to epithelial cell damage, alveolar macrophages secrete chemokines to recruit neutrophils into the lung, triggering an inflammatory cascade that exacerbates lung injury [5]. Thus, alveolar macrophages play a pivotal role in regulating immune homeostasis in acute lung injury.

Lipopolysaccharide (LPS) is a distinctive component that forms an integral part of the cell walls of Gram-negative bacteria [6]. As an endotoxin, LPS is frequently utilized to establish a sepsis model of acute lung injury in mice [7]. LPS has the ability to activate Toll-like receptor 4 (TLR4), which is part of the Toll-like receptor protein family that identifies common pathogen-associated molecular patterns (PAMPs), thus activating diverse pro-inflammatory signaling pathways including NF-κB, ERK, and MAPKs, leading to heightened production of pro-inflammatory factors such as (IL-1β, IL-6, TNF-α, etc.) [8]. Macrophages express a variety of TLRs on their surface, enabling a rapid response to LPS stimulation signals for immune defense purposes [9].

LncRNAs, or long non-coding RNAs, are a category of RNA molecules characterized by a length exceeding 200 nucleotides [10]. They lack open reading frame (ORF) and have no protein-coding function. In general, lncRNAs predominantly regulate gene expression across three distinct levels: epigenetic, transcriptional, and post-transcriptional mechanisms [11]. Many studies have underscored the regulatory influence of lncRNAs in lung diseases. For instance, lncRNA ZNRD1-AS1 inhibits tumor growth through the suppression of lung cancer cell proliferation, migration, and angiogenesis [12]. LncRNA HCG18 loaded by polymorphonuclear neutrophil-secreted exosomes plays a key role in regulating macrophage polarization and ALI [13]. The lncRNA GAS5 modulates phagocytosis and chemotaxis of macrophages treated with MIP [14]. LncRNA Gm26917, a rarely studied LncRNA, possesses functional roles that remain incompletely characterized. According to reported studies, lncRNA Gm26917 involved with the regulation of cell proliferation, inflammation, oxidative stress, and viral infection [15–17]. Our investigation also revealed that lncRNA Gm26917 modulates macrophage inflammatory responses in LPS-induced acute liver injury [18]. However, the specific role of lncRNA Gm26917 in acute lung injury remains unexplored.

NKRF (NF kappa B repressing factor), is also known by its alternative designations, transcription factor NRF or protein ITBA4, and is a widely and constitutively expressed negatively regulated transcription factor [19]. It has been reported that the NKRF binds directly to the promoter of IFN-β, IL-8 and NOS2 to repress their basal transcription [20–22]. Additionally, it binds to specific negative regulatory elements (NREs) and interacts directly with NF-κB through its N-terminus, thereby specifically inhibiting the transcriptional activity of NF-κB proteins [23]. Moreover, it has been reported that WWP2 can promote the ubiquitination of NKRF and exacerbate the progression of acute myeloid leukemia (AML) [24]. Five transcription factors belong to the NF-κB family in mammals: p50, p52, RelA (p65), RelB, and c-Rel. p50 and p65 form the predominant heterodimer (p50/p65) in the NF-κB signaling pathway [25, 26]. Upon stimulation by inflammatory signals, the IKK complex in the cytoplasm becomes activated, facilitating the phosphorylation and degradation of IκBα. The dissociation of IκBα allows for the translocation of the p50/p65 heterodimer to the nucleus and binding to specific gene promoters, thereby regulating the expression of proteins that promote and inhibit inflammation [27–29]. Consequently, hindering the NF-κB signaling pathway attenuates LPS-induced acute lung injury.

In this study, we investigated the roles of lncRNA Gm26917 in mouse alveolar macrophages and acute lung injury. First, qRT-PCR showed that Gm26917 was significantly highly overexpressed in ALI and LPS-treated mouse alveolar macrophages. Furthermore, silencing of Gm26917 attenuated macrophage inflammatory response, modulated their function, and alleviated LPS-induced inflammation and pathological injury in mouse lungs. Additionally, silencing Gm26917 reduced NKRF ubiquitination, facilitating NKRF binding to p50 and suppressing the production of inflammatory factors downstream of NF-κB pathway. In summary, this study comprehensively elucidates the mechanism by which Gm26917 roles critically in LPS-induced alveolar macrophages and acute lung injury.

Materials and Methods

Mice

Male C57BL/6 mice, aged 6 to 8 weeks, were obtained from the Experimental Animal Center at Anhui Medical University. The animals were housed in a designated pathogen-free environment and given access to standard food and fresh water. The ambient temperature was maintained at 24 ± 2 °C, humidity at 40% −70%, and a 12-h light and 12-h dark cycle was employed. All animal protocols received approval from the Animal Care and Use Committee at Anhui Medical University.

Establishment of ALI Mouse Model

C57BL/6 mice were acclimatised for one week and injected with lentivirus (LV-sh-NC or LV-sh-Gm26917, 1 × 109 TU/ml) into the tail vein, and the viral infiltration time was 72 h. After 72 h, LPS (Escherichia coli O111: B4, Sigma-Aldrich, St. Louis, MO) was added for stimulation to establish an acute lung injury model. The mice were initially anesthetized through an intraperitoneal injection of a 10% chloral hydrate solution (3.5 ml/kg, Phygene Biotechnology Co, Ltd) and then stimulated with LPS (10 mg/kg) by intratracheal instillation. The control group was administered an equivalent volume of PBS. After administration, the mice were placed vertically and shaken slowly for 1 min to distribute the LPS or PBS evenly in the left and right lungs of the mice. After 12 h, the mice were sacrificed and the whole left lung was harvested for W/D ratio measurement. The upper lobe of the right lung was embedded in paraffin and the remaining lung tissues were preserved at −80 °C.

Lung Wet to Dry (W/D) Ratio

The entire left lung of the mice was removed and the wet weight was weighed after PBS washing. The lung tissue was then freeze-dried and the dry weight was weighed. Finally, pulmonary oedema in the mice was scored according to the wet weight/dry weight ratio.

Histopathological Analysis

Lung tissues were removed from the sacrificed mice and immersed in 4% paraformaldehyde. They were then embedded in paraffin. The paraffin sections were dewaxed and stained with eosin and haematoxylin. Finally, the stained sections were dehydrated in ethanol, cleared in xylene and sealed in neutral gum. Pathological damage to the lung tissue was observed under the microscope.

Immunohistochemical Analysis

First, the embedded paraffin sections were dewaxed, and the lung tissue was treated with sodium citrate repair solution, and then 3% hydrogen peroxide was used to inactivate the endogenous peroxidase. Next, 10% goat serum was added to block the sections and apply the antibody. After overnight, the sections were stained with DAB solution and hematoxylin nuclear staining. Finally, the images were observed through a microscope and analyzed. Five fields of view were observed for each sample and the number of positive cells was counted.

Cell Culture, Transfection, and LPS Treatment

The MH-S cell line was purchased from Fuheng Biology (Shanghai, China). MH-S cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and grown in a 37 °C incubator containing 95% air and 5% CO₂.

The transfection of siRNA and plasmid was carried out according to the instruction manual of jetPRIME ® transfection reagent (Polyplus). The siRNA was ordered from General Biosystems Co., LTD (Anhui, China), and the plasmid was designed and synthesized by Miaolingbio Bioscience & Technology Co., LTD (Wuhan, China). The siRNA sequence is: si-Gm26917(5’-CAAAACCAACCCGGUGAGC-3’), si-NKRF(5’-AGACAAAUCCUGAGUAUAU-3’).

After 52 h of siRNA transfection, fresh medium was replaced and LPS solution was added to achieve a stimulation concentration of LPS of 800 ng/ml. Cells were collected for qPCR and Western blot analysis after 12 h of LPS stimulation.

RNA Extraction and qRT-PCR

Total RNA of cells and tissues was extracted using a rapid RNA extraction kit (ES-RN001, Yishan Biotechnology Co., LTD). The cDNA was synthesized according to the instructions of Hifair ® II 1st Strand cDNA Synthesis Kit (11150ES60, YEASEN).

Real-time quantitative PCR was performed using Hieff ® qPCR SYBR Green Master Mix (11201ES08, YEASEN) to detect relative gene expression and CFX96 Real-Time PCR System (CFX96, Bio-Rad, USA) was used for detection. The expression levels were determined using the comparative Ct method according to Formula^2−∆∆Ct, with the expression of each gene normalized to Gapdh.

Cell Viability Assay (CCK-8)

The cultured cells were digested, centrifuged, and counted, and then inoculated into 96-well cell culture plates with approximately 4000 cells per well. The incubation was conducted at a temperature of 37 degrees Celsius, with 5% carbon dioxide and a humidity level of 90%. Each well was added with 10 μl of thawed CCK-8 solution (TargetMol) and cultured for 2 h. Cell activity was assessed by measuring absorbance values at 450 nm using a microplate reader.

Flow Cytometry

Apoptosis was detected by flow cytometry (BD FACSCelestaTM Cell Analyzer) according to the instructions of Annexin V-FITC/PI Double Staining Apoptosis Detection Kit (Bestbio). Cells were harvested and subjected to two washing steps using cold PBS and then the sample was resuspended in 1 × Annexin V binding solution. Annexin V-FITC and PI staining solutions were added successively, gently mixed, and then incubated for 15 min at 2–8 °C in the dark. Immediate detection of apoptotic cells by flow cytometry.

RNA Fluorescence In Situ Hybridization (FISH)

The probe for LncRNA Gm26917 was designed and synthesized by GenePharma (Shanghai, China). The probe sequence is: 5 ' -AGCCCGAGGAGGCGACACCGACACGAGAGAGAAGCGGAGGCGGAGGACGAACCCTCTCCGACACCTCTCTTATCCGC-3'. FISH experiments were conducted following the guidelines provided in the RNA FISH Kit SA-Biotin system (paraffin sections). Firstly, paraffin sections embedded with lung tissue were dewaxed, and then 100 μl of Proteinase K working solution was added to each section and incubated at 37 °C for 30 min. The sections were sealed and rinsed three times at room temperature. To each section, 100 µl of pre-heated denaturation solution was added and incubated at 78 °C for 8 min. The probe powder was prepared in DEPC water as a storage solution with a final concentration of 100 μM, and then the probe working solution was prepared. The storage solution was diluted to 1 μM and denatured in a water bath at 75 °C for 10 min. Then it was added proportionally with SA-Cy3 to PBS (1:1:8) and incubated at 37 °C for 30 min. The prepared probe working solution was mixed with the hybridization buffer and added to the denatured section, then placed in a wet box and incubated at 37 °C for 12–16 h. For combined immunofluorescence staining, the incubated sections were washed three times with PBS and treated with CD68 (ab125212) antibody for 2 h at 37 °C. After washing with PBS for three times, fluorescently labelled secondary antibody (ab150080) was added and incubated for 1 h at 37 °C. Finally, the anti-quenching agent containing DAPI was added, and the image was observed with a fluorescence microscope.

RIP (RNA Binding Protein Immunoprecipitation)

Cells were exposed to formaldehyde at a final concentration of 0.3% for a duration of 10 min at 37 °C. Subsequently, the reaction was halted by the addition of 1 ml of glycine, achieving a final concentration of 125 mM, at room temperature. The cells were then harvested, subjected to two washes with cold PBS, and centrifuged. Then RIPA Lysis Buffer (Medium, biosharp) was introduced to completely lyse the cells while maintained on ice. The whole-cell protein lysate was then acquired through centrifugation at 12,000 rpm for a duration of 10 min, divided into two portions and added with IgG and NKRF antibodies, respectively, and incubated on a rotary mixer at 4 °C for 4 h. BeyoMagTM Protein G magnetic beads were pre-washed 3 times with RIPA lysis buffer, and then co-incubated with the above protein-antibody mixture overnight. On the second day, the magnetic beads were washed three times with RIPA buffer, a portion of the beads were eluted with 1 × SDS for subsequent Westen blot experiments, and the remaining portion was supplemented with 0.5 ml of TRIzol reagent for the following qRT-PCR analysis.

RNA-seq Analyses

We extracted total RNA from MH-S cells in the si-NC group and the si-Gm26917 group stimulated with LPS for 12 h. The generated cDNA library underwent sequencing with the Illumina Novaseq6000 platform, conducted by Gene Denovo Biotechnology Co. (Guangzhou, China). In order to obtain high-quality clean reads, the sequencing data were subsequently refined using fastp (version 0.18.0) [30] for additional filtering. The mapped reads from each sample were assembled using a reference-based approach with StringTie version 1.3.1 [31, 32]. For each transcriptional region, the expression abundance and variability were quantified by calculating FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values using RSEM (RNA-Seq by Expectation–Maximization) software [33]. Differential expression analysis of RNAs was conducted between two distinct groups utilizing DESeq2 software [34]. Genes were deemed differentially expressed (DEGs) if they met the criteria of a false discovery rate (FDR) below 0.05 and an absolute fold change greater than 2. Differentially expressed genes were analyzed for GO/KEGG enrichment, and terms with a corrected p-value of 0.05 were deemed significantly enriched among the DEGs.

Lentiviruses, Plasmids and Antibodies

The recombinant lentivirus was synthesized by GenePharma Co., Ltd. (Shanghai, China), and the lentivirus (LV-sh-Gm26917) targeting Gm26917 silencing expression was synthesized according to the siRNA sequence.

Clone pcDNA-Flag-NKRF into a pcDNA3.1 vector containing the CMV promoter and the appropriate epitope tag. Transformation and amplification of the plasmid was performed according to the manufacturer's instructions.

The antibodies used for Western blotting, co-immunoprecipitation or immunofluorescence are as follows: NKRF (DF8755,Affinity), GAPDH (T004,Affinity), His-Tag antibody (66,005–1-Ig, Proteintech), Flag-Tag antibody (66,008–4-Ig, Proteintech), IgG(A7028,Beyotime), p65 (YM3111,Immunoway), p-p65 (YP0191, Immunoway), IKBα (10,268–1-AP, Proteintech), p-IKBα (82,349–1-RR, Proteintech), p50 (66,992–1-Ig, Proteintech), F4 / 80 (sc377009,SantaCruz), CD68 (Ab125212,Abcam).

Statistical Analysis

Data were analyzed using GraphPad Prism 8.0.2 software. Western blots were quantified using ImageJ software. Data values are reported as mean ± SD of at least three independent samples. For comparisons between two groups, Student's t-test was employed, while differences among multiple groups were assessed using one-way ANOVA. A p-value of less than 0.05 was considered statistically significant.

Results

LncRNA Gm26917 is Significantly Upregulated in ALI and LPS-treated MH-S Cells

To explore the expression of lncRNA Gm26917 in acute lung injury, we successfully established a mouse model of acute lung injury based on previous reports [35]. Hematoxylin–eosin staining of mouse lung tissues revealed significant pathological changes in the lungs of mice given LPS stimulation compared to control mice: alveolar wall thickening, hemorrhage, inflammatory cell infiltration, etc. (Fig. 1a). Following LPS stimulation, the lung W/D ratio significantly increased compared to controls, indicating LPS-induced pulmonary edema in mice (Fig. 1b). Additionally, we also analyzed the mRNA levels of inflammatory factors in the lung tissue of mice to determine the impact of LPS stimulation. As depicted in Fig. 1c, the mRNA levels of IL-1, IL-6, and TNF-α exhibited a significant increase following stimulation with LPS. Under these conditions, we found that lncRNA Gm26917 expression in mouse lung tissue was significantly upregulated following LPS treatment compared with controls (Fig. 1d). Concurrently, we induced sepsis in mice via cecal ligation, which yielded comparable results regarding Gm26917 expression in the lungs of septic mice (Fig. 1e, f).

Fig. 1.

LncRNA Gm26917 was highly expressed in ALI and LPS-treated MH-S cells. (a) HE staining to observe the pathological damage of lung tissue in mice before and after LPS treatment. (b) Wet/dry ratio of mouse lungs before and after LPS treatment. (c) RT-PCR was performed to detect the mRNA levels of cytokines (IL-1β, IL-6, TNF-α) in mouse lung tissue before and after LPS treatment. The control group was given the same volume of PBS. (d) The mRNA level of Gm26917 in mouse lung tissues before and after LPS treatment. (e) The mRNA level of Gm26917 in lung tissue of mice in sham operation group and perforated cecum ligation (CLP) model. (f) The mRNA levels of cytokines (IL-1β, IL-6, TNF-α) in mouse lung tissue mice in sham operation group and perforated cecum ligation (CLP) model. (g) For colocalization analysis, lung tissues were costained with Cy3-labeled anti-Gm26917 probe(red) and anti-CD68 (macrophage marker, green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (h) The mRNA levels of cytokines (IL-1β, IL-6, TNF-α) in MH-S cells before and after 800 ng / ml LPS stimulation. (i) The mRNA level of Gm26917 in MH-S cells (mouse alveolar macrophages) stimulated by different concentrations of LPS. (j) The mRNA levels of Gm26917 in MH-S cells stimulated with 800 ng/ml of LPS for a continuous period of time. Data are shown as means ± SD of at least three independent experiments. ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001

Alveolar macrophages, intrinsic immune cells of the lungs, have been reported to play an indispensable regulatory role in the presence of invading pathogenic microorganisms [36]. Therefore, we hypothesized that Gm26917 collaborates with alveolar macrophages to combat external stimuli. FISH immunolocalization revealed co-localization of probe-labeled Gm26917 (red) with CD68-labeled macrophages (green) in mouse lung tissue, confirming Gm26917 localization in alveolar macrophages (Fig. 1g). Next, to further validate this hypothesis, we stimulated MH-S cells with different concentrations of LPS. We found that the mRNA levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) increased sequentially with rising concentrations of LPS (Fig. 1h). Similarly, Gm26917 expression exhibited consistent upregulation in response to increased LPS concentrations (Fig. 1i). This reflects that the expression of Gm26917 is positively correlated with the severity of inflammation. In addition, we observed that the expression of Gm26917 increased with prolonged stimulation duration (Fig. 1j). In conclusion, these findings indicate that Gm26917 expression is significantly up-regulated in LPS-induced MH-S cells and ALI. Meanwhile, these findings underscore the potential involvement of Gm26917 in ALI, especially in alveolar macrophages.

Lentivirus-mediated Silencing of lncRNA Gm26917 Protects Against LPS-induced ALI

Next, to delve deeper into the role of Gm26917 in ALI, mice with silenced Gm26917 expression were generated via tail vein injection of lentivirus (LV-sh-Gm26917). ALI was induced through the intratracheal instillation of LPS, and the efficiency of silencing before and after LPS stimulation is depicted in Fig. 2a. Examination of the lung W/D ratio revealed that LPS-treated Gm26917-silenced mice showed significant improvement in pulmonary edema (Fig. 2b). Furthermore, Gm26917 knockdown, regardless of LPS stimulation, resulted in a reduction of pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α, while simultaneously leading to an elevation in the levels of the anti-inflammatory cytokine IL-10 within the lung tissue of mice (Fig. 2c, d). Concurrently, lung histopathological examination revealed a marked reduction in lung histopathological damage and a decrease in the lung damage score index in mice with silenced Gm26917 expression (Fig. 2e upper, f). Moreover, immunohistochemical staining showed that the amount of macrophage infiltration in the lungs of mice in the Gm26917 knockdown group was also markedly decreased (Fig. 2e lower, g). In summary, silencing of Gm26917 attenuated LPS-induced pulmonary edema and effectively mitigated lung inflammation in mice, indicating a protective effect against the acute lung injury induced by LPS.

Fig. 2.

Knockdown of LncRNA Gm26917 by lentivirus alleviate LPS-induced ALI. (a) Lentivirus infection efficiency at 72 h after lentivirus administration and LPS stimulation for 12 h. (b) Lung W/D ratio 72 h after administration of lentivirus and LPS stimulation for 12 h. (c) Levels of cytokines (IL-1β, IL-6, TNF-α and IL-10) in lungs of mice infected with lentivirus for 72 h. (d) Levels of cytokines (IL-1β, IL-6, TNF-α and IL-10) in the lung of mice after 72 h of lentivirus infection followed by LPS stimulation for 12 h. (e) Histopathology in lung of LV-sh-NC or LV-sh-Gm26917 treated mice 12 h after LPS challenge. Upper panel: Hematoxylin and eosin staining; Lower panel: Immunohistochemical staining for macrophage marker F4/80. (f) Quantitative analysis of lung injury score. (g) Quantitative analysis of F4/80 positive cells. Data are mean ± SD and are representative of three independent experiments. ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001

Knockdown of LncRNA Gm26917 Attenuates LPS-induced Inflammatory Response in MH-S Cells and Modulates their Function

To elucidate the specific role of Gm26917 in alveolar macrophages, we employed siRNA to knock down Gm26917 in MH-S cells and subsequently stimulated the cells with LPS for 12 h. Then, RNA-seq was used to analyze the different gene expression. Volcano plot analysis of significantly differentiated genes between the control and si-Gm26917 groups was performed, identifying 362 up-regulated genes and 141 down-regulated genes in si-Gm26917 group compared with the control group (Fig. 3a). Additionally, we assessed the primary biological functions associated with the differentially expressed genes through Gene Ontology (GO) functional enrichment analysis. This analysis indicated that the knockdown of Gm26917 influenced biological processes including the inflammatory response, modulation of responses to stimuli, and stress in alveolar macrophages (Fig. 3b). Taken together, these analyses indicate that Gm26917 is essential for mediating LPS-induced inflammation within alveolar macrophages.

Fig. 3.

Knockdown of LncRNA Gm26917 attenuates LPS-induced inflammatory responses in MH-S and regulate its function. (a) Differential gene expression data are presented as volcano plots between si-NC-treated cells and si-Gm26917-treated MH-S cells with three repeats. Red and blue points correspond to 2.0-fold changes in the two groups and indicate a FDR < 0.05. (b) Gene ontology analysis of all expression-altered genes and the biological processes they are involved in. (c) The effect of Gm26917 knockdown on the expression of inflammatory cytokines in MH-S cells before and after LPS treatment. (d) The effect of Gm26917 knockdown on the expression of chemokines in MH-S cells before and after LPS treatment. (e) The effect of Gm26917 knockdown on the expression of phagocytosis-related genes in MH-S cells before and after LPS treatment. (f) CCK-8 was used to detect the effect of Gm26917 knockdown on the viability of MH-S cells before and after LPS treatment. Measurement of absorbance values at 450 nm. (g) Flow cytometry analysis of the effect of Gm26917 knockdown on apoptosis in MH-S cells before and after LPS treatment. (h) Quantitative analysis of apoptosis rates. Data represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

To further substantiate the previous analysis, we evaluated the expression levels of mRNA associated with inflammatory factors, chemokines, and phagocytosis-related genes using qRT-PCR. We observed that the knockdown of Gm26917 markedly inhibited the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-12β, while concurrently enhancing the expression of the anti-inflammatory cytokine IL-10 both before and after LPS stimulation (Fig. 3c). The expression of chemokines (CCL2, CCL4, CXCL2, CXCL10) was also significantly diminished in the si-Gm26917 group, whereas the expression of CCL24 was increased (Fig. 3d). CCL2, CCL4, CXCL2, and CXCL10, known as pro-inflammatory chemokines, are capable of recruiting immune cells to sites of inflammation upon LPS stimulation. This implies that Gm26917 knockdown may inhibit the secretion of pro-inflammatory cytokines by macrophages. It has been reported that CCL24 is regulated by M2 macrophage polarization [37], could be associated with the anti-inflammatory effect of Gm26917 knockdown, potentially via the promotion of macrophage polarization toward the M2 phenotype. Meanwhile, the expression of phagocytosis-related genes was significantly increased following Gm26917 knockdown (Fig. 3e), which further demonstrated that Gm26917 silencing serves to protect against inflammation by augmenting phagocytosis in macrophages.

In addition, we evaluated the impact of Gm26917 on macrophage activity by CCK-8 assay. The findings indicated that silencing Gm26917 markedly decreased cell viability after 48 h (Fig. 3f). Meanwhile, flow cytometry analysis of cell apoptosis revealed that Gm26917 knockdown significantly reduced cell mortality both prior to and following LPS treatment (Fig. 3g, h).

Collectively, these findings underscore the protective role of Gm26917 in LPS-induced MH-S cells, identifying it as a crucial regulatory factor in alveolar macrophages.

LncRNA Gm26917 Interacts with NKRF and the Knockdown of Gm26917 Promotes NKRF Protein Expression

Although lncRNAs do not encode proteins, they often interact with RNA-binding proteins (RBPs) to execute various biological functions [38]. We employed the catRAPID tool to predict RNA-binding proteins that interact with Gm26917. NKRF was identified as having a strong binding affinity for Gm26917. Subsequently, we investigated the RNA binding region of NKRF and found that the total RNA interaction score for NKRF was 0.62, with the binding site located at the C-terminus of the protein (Fig. 4a). Then, to elucidate the detailed binding interactions between Gm26917 and NKRF, we conducted an interaction analysis using the catRAPID fragment tool. The interaction matrix was displayed as a heatmap. The binding region of NKRF and Gm26917 was predominantly localized between the C-terminus of the protein and the 1000 nt to 3000 nt regions of RNA (Fig. 4b).

Fig. 4.

Gm26917 interacts with NKRF, and knockdown of Gm26917 promotes NKRF protein expression. (a) Prediction of RNA binding probability of RNA-binding protein NKRF by catRAPID analysis. (b) The interaction tendency between NKRF and lncRNA Gm26917 was predicted by catRAPID analysis. (c) RNA immunoprecipitation (RIP) assay was performed to determine the enrichment of Gm26917 on NKRF protein in MH-S cells. Western blot analysis of immunoprecipitated NKRF. (d) Knockdown of Gm26917 before and after LPS treatment did not affect the mRNA level of NKRF in MH-S cells. (e) Knockdown of Gm26917 before and after LPS treatment did not affect the mRNA level of NKRF in lung tissue of mice. (f) Knockdown of Gm26917 increased the content of NKRF protein in MH-S cells and mouse lung tissue. Upper: MH-S cells were transfected with si-NC (lane 1–3) or si-Gm26917 (lane 4–6). NKRF expression in the control and si-Gm26917 groups was examined by Western blotting. Lower: Mouse lung tissue infected with lentivirus (LV-sh-NC and LV-sh-Gm26917). NKRF expression in the LV-sh-NC and LV-sh-Gm26917 groups was examined by Western blotting. GAPDH served as a loading control. (g) After LPS treatment, knockdown of Gm26917 increased the content of NKRF protein in MH-S cells and mouse lung tissue. Western blot analysis of NKRF expression in each treatment group. GAPDH served as a loading control. (h) Immunofluorescence (IF) was used to analyze the effect of knockdown of Gm26917 on the expression of NKRF in MH-S cells before and after LPS treatment. Variables are presented as mean ± SD. ns, non-significant, *P < 0.05; **P < 0.01; ***P < 0.001

To validate these predictions, we used RNA immunoprecipitation (RIP) to ascertain whether Gm26917 interacts with NKRF. Whole cell lysates were collected and performed immunoprecipitation (IP) using an NKRF antibody. Subsequently, total RNA from the precipitate was extracted and reverse transcribed. qRT-PCR was conducted utilizing specific primers for Gm26917 to assess whether Gm26917 was enriched in the precipitate. We observed that, compared with the IgG control group, Gm26917 was markedly enriched in the NKRF precipitate (Fig. 4c, above). Western blot was employed to verify the pull-down efficiency of NKRF (Fig. 4c, below). Consistent with the predicted results, our data confirm that Gm26917 interacts with NKRF.

To further investigate the relationship between Gm26917 and NKRF, we knocked down Gm26917 to observe the changes in NKRF. The results demonstrated that LPS stimulation resulted in a marked reduction in the mRNA levels of NKRF. Interestingly, however, knockdown of Gm26917 in both MH-S cells and mice did not influence the mRNA levels of NKRF (Fig. 4d, e). Additionally, we investigated the impact of Gm26917 on NKRF protein levels. Western blot analysis revealed that Gm26917 knockdown significantly increase the protein levels of NKRF (Fig. 4f, g). Consistent with the Western blot findings, immunofluorescence microscopy confirmed that NKRF localized in the nucleus, with its expression significantly diminished following LPS treatment. Conversely, Gm26917 knockdown was observed to enhance the expression of NKRF before and after LPS stimulation (Fig. 4h). These results suggest that Gm26917 may regulate the stability of NKRF protein at the translational level.

Silencing of Gm26917 Inhibits NF-κB Signaling Pathway by Reducing the Ubiquitination of NKRF and Enhancing the Binding of NKRF to p50

Post-translational modifications (PTMs) are crucial mechanisms for increasing protein diversity. Common PTMs include phosphorylation, acetylation, and ubiquitination [39]. Many studies have demonstrated that lncRNAs can influence protein function through post-translational modifications [40]. Consequently, we hypothesized that Gm26917 regulates the stability of NKRF by modulating its ubiquitination.

To test this hypothesis, we co-transfected flag-NKRF and His-tagged ubiquitin into 293 T cells, establishing both control and Gm26917 knockdown groups.MG-132 was added 6 h before cell collection to inhibit protein degradation. Whole-cell lysates were immunoprecipitated with a flag antibody and immunoblotted with a His antibody. The results showed that siRNA-mediated knockdown of Gm26917 significantly reduced the ubiquitination level of NKRF (Fig. 5a). To enhance the reliability of the results, a reverse validation experiment was conducted. Whole-cell lysates were immunoprecipitated with a His antibody and subsequently immunoblotted with an NKRF antibody. Similarly, the binding between NKRF and ubiquitin was significantly reduced after the knockdown of Gm26917 (Fig. 5b).

Fig. 5.

Knockdown of Gm26917 inhibits NF-κB signaling pathway by inhibiting the ubiquitination of NKRF and increasing the binding of NKRF to p50. (a) Knockdown of Gm26917 significantly reduced the ubiquitination of NKRF. 293 T cells expressed NKRF-flag with si-NC or si-Gm26917. Upper: Lysates were immunoprecipitated (IP) with anti-flag antibodies followed by detection with anti-His antibodies or NKRF antibodies by Western blotting. Lower: The input protein NKRF-flag and loading control (GAPDH) were detected by Western blotting. (b) Upper: Lysates were immunoprecipitated (IP) with anti-His antibodies followed by detection with NKRF antibodies or anti-His antibodies by Western blotting. Lower: The input protein NKRF-flag and loading control (GAPDH) were detected by Western blotting. (c) The top 20 signaling pathways enriched by differentially expressed genes (DEGs) were analyzed by KEGG pathway analysis. (d) Knockdown of Gm26917 inhibited NF-κB signaling pathway. Western blot was used to analyze the effect of knockdown of Gm26917 on phosphorylated p65 and IKBα in MH-S cells. (e) Quantitative analysis of phosphorylated p65 and IKBα in MH-S cells. (f) Knockdown of Gm26917 increased the binding of NKRF to p50. Immunoblotting analysis of NKRF, p65, and p50 in co-immunoprecipitation (Co-IP) experiments. MH-S cells expressed NKRF-flag with si-NC or si-Gm26917. Data are the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

RNA transcriptome sequencing analysis identified the top 20 KEGG-enriched pathways associated with Gm26917 under LPS induction (Fig. 5c). We specifically focused on the NF-κB signaling pathway for Toll-like receptor signaling cascade. The role of Gm26917 in the NF-κB signaling pathway was assessed by evaluating changes in NF-κB protein levels, including IKBα, p-IKBα, p65, and p-p65. The results demonstrated that knocking down Gm26917 significantly reduced the phosphorylation level of NF-κB protein (Fig. 5d, e). This suggests that knockdown of Gm26917 shows anti-inflammatory effects by inhibiting the NF-κB signaling pathway.

We aim to further elucidate the mechanisms by which Gm26917 and NKRF modulate the NF-κB signaling pathway. A recent study reported that NKRF binds to p50 and inhibits the NF-κB signaling pathway [41]. NKRF was immunoprecipitated using Flag magnetic beads, followed by Western blotting to detect p65 and p50 proteins. Co-immunoprecipitation (Co-IP) experiments revealed that NKRF interacts with p50 but not p65, and that knockdown of Gm26917 enhances the binding of NKRF to p50 (Fig. 5f). These findings indicate that silencing of Gm2691 inhibits the NF-κB signaling pathway by reducing the ubiquitination of NKRF and increasing the binding of NKRF to p50.

Gm26917 and NKRF Co-regulate LPS-induced Inflammatory Responses in Macrophages

Next, to study the role of Gm26917 and NKRF in LPS-induced macrophage inflammation, we overexpressed NKRF in MH-S cells (Fig. 6a). The overexpression of NKRF in MH-S cells led to a significant decrease in the mRNA levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α, both before and after LPS stimulation, while the mRNA level of the anti-inflammatory factor IL-10 was markedly increased (Fig. 6b). Moreover, we co-transfected MH-S cells with si-Gm26917 and an NKRF plasmid, followed by LPS treatment. The mRNA level of NKRF is shown in Fig. 6c. The inflammatory cytokine results demonstrated that NKRF overexpression, in the context of Gm26917 knockdown, further inhibited inflammation both before and after LPS treatment (Fig. 6d).

Fig. 6.

Gm26917 and NKRF co-regulate LPS-induced inflammatory responses in macrophages. (a) The level of NKRF mRNA was detected by qRT-PCR. NKRF-flag plasmid was transfected into MH-S cells before and after LPS stimulation. (b) The mRNA levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and anti-inflammatory factors (IL-10) were analyzed by qRT-PCR. NKRF-flag plasmid was transfected into MH-S cells before and after LPS stimulation. (c) The mRNA level of NKRF was analyzed by qRT-PCR. Before and after LPS stimulation, si-Gm26917 and NKRF-flag plasmids were co-transfected into MH-S cells. (d) Knockdown of Gm26917 and overexpression of NKRF can further reduce the levels of inflammatory factors before and after LPS treatment. The mRNA levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and anti-inflammatory factors (IL-10) were analyzed by qRT-PCR. (e) The level of NKRF mRNA was detected by qRT-PCR. si-NC and si-NKRF were transfected into MH-S cells before and after LPS stimulation. (f) The mRNA levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and anti-inflammatory factors (IL-10) were analyzed by qRT-PCR. si-NC and si-NKRF were transfected into MH-S cells before and after LPS stimulation. (g) The mRNA level of NKRF was analyzed by qRT-PCR. Before and after LPS stimulation, si-Gm26917 and si-NKRF were co-transfected into MH-S cells. (h) Knockdown of Gm26917 while knocking down NKRF can partially rescue the decreased levels of inflammatory factors. The mRNA levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and anti-inflammatory factors (IL-10) were analyzed by qRT-PCR. Data represent the mean ± SD of three independent experiments. ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001

Conversely, siRNA-mediated knocking down NKRF in MH-S cells (Fig. 6e) exacerbated the LPS-induced inflammatory response (Fig. 6f). Following the same approach, we co-transfected MH-S cells with si-Gm26917 and si-NKRF, followed by LPS treatment. The mRNA level of NKRF following this treatment is shown in Fig. 6g. The results indicated that silencing NKRF, in conjunction with Gm26917 knockdown, partially restored inflammatory cytokine levels that were diminished by si-Gm26917, both before and after LPS stimulation (Fig. 6h).

In conclusion, these findings suggest that Gm26917 and NKRF exert a synergistic effect in LPS-induced inflammatory response, and they may be potential targets for the treatment of LPS-induced ALI.

Discussion

Alveolar macrophages, as the first line of immune defense in the lung, are crucial in maintaining immune homeostasis during acute lung injury [42]. Our study demonstrated that lncRNA Gm26917 is markedly overexpressed in both ALI and LPS-treated alveolar macrophages. Silencing Gm26917 via lentivirus-mediated methods effectively mitigated acute lung injury induced by LPS. Furthermore, knocking down Gm26917 significantly attenuates the LPS-induced inflammatory response in alveolar macrophages. Additionally, we observed an interaction between Gm26917 and NKRF. Knockdown of Gm26917 inhibited the ubiquitination of NKRF, thereby enhancing NKRF binding to p50 and further obstructing the NF-κB signaling pathway (Fig. 7). In this study, we present a comprehensive analysis of the function of Gm26917 in alveolar macrophages during acute lung injury.

Fig. 7.

Schematic representation of the molecular mechanism of Gm26917 in LPS-induced inflammation in ALI. LPS stimulation results in elevated expression levels of Gm26917 in alveolar macrophages, where it subsequently interacts with NKRF to facilitate its ubiquitination. This process inhibits the binding of NKRF to p50, thereby promoting the nuclear translocation of the p65-p50 dimer and subsequently activating the NF-κB signaling pathway. Within this regulatory framework, Gm26917 plays a critical role in mediating the inflammatory response of alveolar macrophages in LPS-induced acute lung injury

Acute lung injury and its severe form acute respiratory distress syndrome (ARDS) are still the leading cause of death in ICUs [43]. Over the past decade, lncRNAs have been a focal point in the research of various diseases [44]. Many studies have demonstrated that lncRNAs are highly expressed in acute lung injury across mouse models or cell lines, and that targeting these lncRNAs can significantly mitigate ALI [45]. For instance, lncRNA TUG1 is downregulated in CLP model mice. Overexpression of TUG1 can target miR-34b-5p and promote GAB1 to improve sepsis-induced acute lung injury and inflammatory response [46]. Moreover, mechanistic and functional studies suggest that lncRNAs may exacerbate the inflammatory response to sepsis by modulating various cellular signaling pathways. Lin et al. reported that knocking down lncRNA MALAT1 markedly ameliorates septic lung injury in mice, mechanistically by inhibiting the up-regulation of p38 in mouse lung tissues thereby blocking the p38 MAPK/p65 NF-κB signaling pathway [47]. Another study on the mechanism of lncRNA NEAT1 showed that inhibition of NEAT1 could inhibit the activation of HMGB1 / RAGE and NF-κB signaling in LPS-induced ALI [48]. In this study, we observed a significant increase in lncRNA Gm26917 expression in LPS-treated MH-S cells. However, in the absence of LPS stimulation, the knockdown of Gm26917 did not lead to a significant improvement in the lung W/D ratio, lung pathological damage, or macrophage infiltration. This may be attributed to the more pronounced effect of Gm26917 in the inflammatory state, whereas its effect is less apparent under normal conditions. Furthermore, the knockdown of Gm26917 effectively mitigated LPS-induced acute lung pathological damage and inflammatory responses.

Macrophages are extensively distributed throughout the human body and play a crucial role in the immune defense, immune self-stabilization and immune surveillance of the organism [49]. Alveolar macrophages, serving as the body's primary defense against foreign microorganisms invading the lungs, have been widely reported in studies of sepsis-induced acute lung injury [50]. Xia et al. found that AdMSC-Exos could provide mitochondrial components and improve the integrity of impaired mitochondria in LPS-induced alveolar macrophages, thereby restoring macrophage immune homeostasis and attenuating lung injury [51]. Wang et al. summarized the role of macrophage phenotype and polarization regulation in ALI/ARDS. In the pathogenesis of ALI/ARDS, M1 macrophages are primarily involved in pro-inflammatory responses, chemotaxis, free radical production, and the clearance of pathogenic microorganisms, while M2 macrophages mediate anti-inflammatory effects and facilitate tissue remodeling and lung repair [52]. Our findings indicate that lncRNA Gm26917 regulates multiple functions of MH-S cells. Knockdown of Gm26917 attenuated LPS-induced macrophage inflammatory responses and reduced the chemotactic ability of M1-type macrophages, while augmenting the chemotactic ability of M2-type macrophages. Additionally, knockdown of Gm26917 influenced macrophage viability and apoptosis.

An intricate regulatory network governs RNA-RBP interactions. Given the many-to-many interactions between proteins and RNAs, it can be inferred that each RBP can bind to multiple RNAs, while each RNA typically engages with several RBPs [53]. This indicates that a lncRNA can engage in various biological processes through its interaction with different RBPs, thereby exerting distinct biological functions. Furthermore, it is plausible that lncRNA interacts with diverse RBPs across various tissues to modulate analogous biological mechanisms. We previously found that lncRNA Gm26917 binds to the N-terminus of ANXA1 and affects the inflammatory response in hepatic macrophages by regulating the ubiquitylation level of ANXA1 [18]. In this study, we utilized bioinformatics tools to predict proteins that interact with Gm26917. NKRF was predicted to bind to Gm26917 with a high binding score. We subsequently validated it through RNA immunoprecipitation. NKRF, as a negative transcription factor, plays a critical role in the regulation of gene expression in cells. Several studies have highlighted the significant role of NKRF in various cancers, including lung, gastric, and pancreatic cancers, among others [54–56]. In addition, NKRF acts as a direct inhibitor of the NF-κB signaling pathway and regulates inflammatory responses induced by LPS stimulation [57–59]. Moreover, it has been shown that NKRF can be modified post-translationally by ubiquitination and methylation [24, 60].

LncRNA is closely associated with proteins, and research has shown that it can functionally make epigenetic modifications to proteins, such as ubiquitination, phosphorylation, and acetylation [61–63]. Similarly, our study revealed that knockdown of Gm26917 inhibited inflammation by reducing ubiquitination of NKRF. In addition, we also found that knockdown of Gm26917 increased the binding of NKRF to p50 by Co-IP assay. This effect may be attributed to NKRF's inhibition of the nuclear translocation of p50, which in turn suppresses the expression of inflammatory factors downstream of the NF-κB pathway. Consequently, we identified a novel mechanism through which the silencing of lncRNA Gm26917 plays a negative regulatory role in the NF-κB inflammatory pathway by targeting and inhibiting NKRF ubiquitination.

In summary, the interaction of Gm26917 with NKRF and p50 regulates LPS-induced inflammatory responses in alveolar macrophages. In this study, knockdown of Gm26917 alleviated LPS-induced ALI, and overexpression of NKRF blocked the NF-κB pathway thereby reducing the production of inflammatory factors. Consequently, targeting Gm26917 via the NKRF/NF-κB axis may have therapeutic potential for acute lung injury.

Author Contribution

Y. Z.: Writing- original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. C. Z. and L. M.: Software, Validation, Methodology, Investigation. X. L.: Investigation, Validation. W. L.: Investigation, Conceptualization. M. S., L. Z. and Y. W.: Methodology, Formal analysis. Q. Z.: Validation, Methodology. M. S.: Validation, Methodology. W. S.: Writing- review and editing, Supervision, Conceptualization.

Funding

This work was supported by the National Natural Science Foundation of China (82071770); Research Level Improvement Project of Anhui Medical University (2021xkjT001); Anhui Provincial Natural Science Foundation (2008085QH371); Scientific Research of BSKY in Anhui Medical University (XJ201601); Research and Practical Innovation Projects of AHMU (YJS20230141).

Data Availability

The datasets utilized in this study are accessible through online repositories. The relevant repository details and accession numbers are as follows: SRA database within NCBI, accession number PRJNA1141158.

Declarations

Ethics Approval

All animal studies received approval from the Animal Care and Use Committee at Anhui Medical University. All animal experimental procedures were conducted in strict compliance with the regulations governing the management of experimental animals established by Anhui Medical University.

Competing Interest

The authors declare no competing interests.