Long Noncoding RNA SNHG4 Remits Lipopolysaccharide‐Engendered In ammatory Lung Damage by Inhibiting METTL3-Mediated m6A Level of STAT2 mRNA

Si-Xiu Li Children's Hospital A liated to Xi'an Jiaotong University Wen Yan Childrens' Hospital A liated to Xi'an Jiaotong University Jian-Ping Liu Children's Hospital A liated to Xi'an Jiaotong University Yu-Juan Zhao Children's Hospital A liated to Xi'an Jiaotong University Lu Chen (  luchenh@21cn.com ) Childrens' Hospital A liated to Xi'an Jiaotong University https://orcid.org/0000-0001-9019-2007


Introduction
Neonatal pneumonia (NP) is one of the ordinary diseases in the neonatal period. It is a vital cause of perinatal death, and its main clinical manifestations include cough and lung ne wet rales [1]. NP is divided into aspiration pneumonia and infectious pneumonia, of which infectious pneumonia accounts for a large proportion [2]. Many factors contribute to the development of infectious neonatal pneumonia, including bacteria, viruses, molds, mycoplasma pneumonia (MP) and other pathogenic factors. The descending infection department caused respiratory infection, the clinical manifestations were no bronchitis, pneumonia and so on. In severe cases, it can accumulate in the brain, heart, liver, and kidney, followed by encephalitis, myocarditis, and hepatitis, among other complications [3]. However, the pathogenesis of neonatal pneumonia is still controversial, and clinical treatment is still di cult. Therefore, it is of great signi cance to explore the pathogenesis of neonatal pneumonia from the molecular level. Lipopolysaccharide (LPS), a major bioactive component of Gram-negative bacteria pathogens, can cause severe in ammatory reactions in the lungs [4]. Therefore, LPS induced in ammatory injury is a commonly used model to study the pathogenesis and treatment of NP.
Emerging evidence suggests that lncRNA is a novel insight in various diseases, such as lung injury. In pneumonia, a potential key lncRNA pro le in peripheral blood has been identi ed by lncRNA sequencing [5]. Several lncRNA were further revealed to be associated with lung in ammation. A study revealed that lncRNA-MIAT inhibits p38 MAPK and NF-κB attenuated LPS induced lung in ammatory responses in mice through a pathway that downregulated miR-15 [6]. Previous studies show a link between SNHG4 and in ammation. Overexpression of SNHG4 cloud inhibit the expression of in ammatory factors in microglia [7]. Unfortunately, to date, the role of SNHG4 in the in ammatory response in pneumonia remains unclear.
N6-methyladenosine (m 6 A) has been reported as the most prevalent internal mRNA modi cation in eukaryotes, and its role in autoimmunity, in ammation and cancer has attracted close attention. The key enzymes for m 6 A methylation modi cation principally include m 6 A methyltransferase (writer), m 6 A demethylase (eraser) and m 6 A RNA-binding protein (reader) [8]. Methyltransferase-like 3 (METTL3) is a key enzyme of m 6 A methylation modi cation and an important member of the methyltransferase complex including METTL3, METTL4, and Wilms tumor 1-associated protein (WTAP) [9]. It has been demonstrated that m 6 A methylation mediated by METTL3 is necessary for in ammatory responses [10].
A research showed that METTL3 de ciency maintained long-chain fatty acid absorption by inhibiting TRAF6-dependent in ammatory responses [11]. Moreover, METTL3 promoted LPS-induced microglial in ammation by activating the TRAF6-NF-κB pathway [12]. Except for 'writers' and 'erasers', the modi cation is recognized and bound by m 6 A 'readers' in mammalian cells, mainly YTHDF1-3 and YTHDC1, 2, which are from YTH-domain family proteins, thus contributing to various biological processes, such as viral infections and tumorigenesis. YTHDF1 (YTH domain family 1) is the most effective m 6 A reader that weakens mRNA stability by recognizing and distributing m 6 A-containing mRNAs to processing bodies [13]. Recent reports mentioned that YTHDF1 could prevent in ammation in cerebral ischemia / reperfusion injury by m 6 A modi cation of p65 mRNA translation [14]. Nevertheless, the role of METTL3-mediated m 6 A / YTHDF1 in NP remains vague up to date. Based on the above reports, we speculated that SNHG4 and METTL3 might be involved in the progression of NP by regulating in ammatory responses. Therefore, in the present study, we examined whether SNHG4 affects LPS induced injury in WI-38 cells by regulating METTL3, and explored the regulatory correlation between METTL3 mediated m 6 A / YTHDF1 and the downstream possible target genes to decode the molecular mechanism by which SNHG4 alleviates lung injury and nd a novel target for the treatment of NP.

Materials And Methods
Page 4/23 2.1 Collection of serum samples Peripheral venous blood (3 ml) was collected from 15 patients (10 males and 5 females) with neonatal pneumonia; Mean age 10-28 days) and 15 healthy volunteers (10 males and 5 females; Mean age 10-28 days) at Xi'an children's hospital. Patients with other complications or previous anti-in ammatory treatment were excluded. Control blood samples were obtained from persons with normal physical examination results. After collection, blood was centrifuged at 2000 rpm, and then the supernatant was obtained as serum sample and stored at -80°C 2.2 Animals and experimental groups Forty-eight one week old CD-1 mice were purchased from experimental animal center of Xi'an Jiaotong University (Xian, China). All mice were housed under a 12 h light/dark cycle with constant temperature about 25°C and relative humidity approximating 55%. The mice had free access to food and water for 10 days prior to the experiment. The mice were randomly divided into four groups of 10 mice each. After 10 days, mice received an intraperitoneal injection of 22 mg / ml sodium pentobarbital (diluted in saline) followed by 167 µM LPS (60 µL) Saline solution was instilled into the oral cavity through the posterior pharyngeal wall. Pinch the nares quickly and hold for 30 seconds, model is successful when all uid is absorbed into the nasal cavity, and slight tracheal rales appear. Lentiviral vectors containing pcDNA-SNHG4 (150 µM) or pcDNA-3.1 were intratracheally injected into mice. Twenty-one days after establishing the model, mice were intraperitoneally injected with 3% sodium pentobarbital and euthanized by overdose anesthesia at a dose of 90 mL / Kg, and organs and tissues were removed for follow-up studies. In addition, macrophages and neutrophils in alveolar lavage uid were collected as previously described. The protocol of this study was approved by the animal care and use Committee of the children's Hospital of Xi'an Jiaotong University.

Cell Lines and Culture
Human lung broblasts (WI-38) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco's Modi ed Eagle Medium (DMEM) (Gibco, Rockville, MD) supplemented with 10% fetal bovine serum (FBS) (HyClone, Salt Lake City, UT) and 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA) at 37°C in a controlled humidi ed atmosphere with 5% CO 2 . 2 × 10 5 cells were plated in 6-well plates and incubated. LPS (Sigma, St. Louis, MO) was diluted by DMEM to 10 ng/mL and the processing time was 6 hr.

Cell transfection
The pcDNA-SNHG4, pcDNA-METTL3, pcDNA-STAT2, pcDNA-YTHDF1 and METTL3 siRNA were all obtained from GenePharma Co., Ltd. (Shanghai, China). Before transfection, WI-38 cells were digested with 1% trypsin treatment. After being counted in a blood counting chamber, the cells were plated onto six-well culture plates for 24 hours and then transfected at 40%-60% con uence. All transfection was performed with Lipofectamine®3000 (Thermo, Waltham, MA, USA) according to manufacturer's instructions. At 48 hours post-transfection, cells were harvested and subjected to next analyses.

Western blotting
The cells were washed twice with ice-cold PBS and lysed by using RIPA lysis buffer (CW Biotech, Beijing, China) supplemented with protease inhibitor (Roche Diagnostics, Basel, Switzerland). Then the protein concentration was measured by BCA protein assay kit (Thermo Fisher Scienti c, Waltham, MA, USA).
Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:3000, Abcam, ab6721) for 1 h at room temperature. β-actin was used as an endogenous control. The bands were visualized by using an ECL Plus Chemiluminescence Reagent Kit (Pierce, Rockford, IL, USA) and were photographed by a chemiluminescence imaging system. Image J software was used to quantify the band densities.

Cell proliferation assay
The Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich, St. Louis, MO, USA) was performed to detect WI-38 cell proliferation. In brief, WI-38 cells were seeded into 96-well microplates. After incubation for 0, 24, 48 and 72 h, 10 µL of CCK-8 solution was added to each well and incubated with HL-1 cells for another 2 h at 37°C in a humidi ed atmosphere with 5% CO 2 . A microplate reader (Molecular Devices, Shanghai, China) was used to measure the absorbance of each well at 450 nm.

Cell apoptosis assay
Annexin V-FITC/PI double staining was used to analyze apoptosis on a ow cytometer (BD Bioscience, San Jose, CA, USA). After 48 h of transfection, the WI-38 cells were detached with EDTA-free trypsin and collected afterwards. The cells were centrifuged for 5 min at 4°C at 1000 rpm, and the supernatant was discarded. Apoptosis was detected by an Annexin V-FITC/PI Apoptosis Detection Kit (Beijing Solarbio Science & Technology, Beijing, China). The WI-38 cells were suspended in a mixture of AnnexinV-FITC and binding buffer (1:40) and incubated at room temperature for 30 min. A mixture of PI and binding buffer (1:40) was then added and shaken, followed by incubation at room temperature for 15 min. Fluorescence was detected by a ow cytometer, and the apoptosis rate was calculated and determined.

Transwell migration assay
The cell motility was detected by treatment with 8.0-µm chamber plates. Firstly, cells were planted into the 8.0 µm chamber plates, then 300 µL of serum-free DMEM medium was added to the upper compartment of the chamber, and 500 µL of DMEM medium supplemented with 10% FBS was added to the lower chamber for 48 h incubation. Then, the non-migratory cells on the upper side of the chamber were suspended with a cotton swab, and then the migratory cells were xed in 4% paraformaldehyde and stained with a crystal violet solution. We stained in ltrating cells using an Olympus IX70 inverted microscope (Olympus Corp, Tokyo, Japan) and randomly selected the best six elds of view, and each experiment was repeated three times.

RNA Immunoprecipitation (RIP)
Total RNA was isolated from WI-38 cells by using Trizol. Anti-m 6 A antibody (Abcam, ab151230) (or anti-METTL3 antibody or anti-YTHDF1 antibody) and anti IgG (Abcam, ab172730) were conjugated to protein A / G magnetic beads in IP buffer (140 mM NaCl, 1% NP-40, 2 mm EDTA, 20 mm Tris pH.7.5) overnight at 4°C. Total RNA was incubated with antibodies in IP buffer, and precipitated RNA was then eluted from the beads. Finally, precipitated RNA and input total RNA were eluted and reverse transcribed for RT-qPCR.
Relative fold enrichment was calculated with the 2 −ΔΔCT method.

Total m 6 A Measurement
Total RNA was isolated by TRIzol (Thermo Fisher, USA) according to the manufacturer's instructions. The relative content of m 6 A in the total RNA was measured by using the EpiQuik m 6 A RNA Methylation Quanti cation Kit (Colorimetric) (P-9005, Epigentek, USA) according to the manufacture's instruction. In brief, 200 ng RNA were administrated with the solution containing the anti-m 6 A antibody. The m 6 A levels were quanti ed by using the colorimetrical analysis via absorbance at 450 nm.

Statistical Analysis
All statistical analyses were performed with the SPSS software (ver.22.0; SPSS, Chicago, IL). All data were shown as mean ± SEM. Student's t-test was performed for the comparison between two groups, and analysis of variance (ANOVA) was performed for comparison among groups. P 0.05 was considered as statistically signi cant difference.

Results
3.1 LncRNA SNHG4 was downregulated in the serum of patients with pneumonia and LPS-induced cell model of pneumonia We examined a total of 30 serum samples including 15 patients with pneumonia and 15 healthy controls. As shown in Fig. 1A, the expression level of SNHG4 was abundantly decreased in the serum from patients with pneumonia compared to the controls. LPS was used to treat human lung broblasts to establish an in vitro cellular model of pneumonia. The dosage of LPS treatment was determined in WI-38 cells. After LPS stimulation (5, 10, and 20 µg / ml) for 24 hours, SNHG4 levels were attenuated with increasing LPS concentrations (Fig. 1B). These results indicated that the downregulated expression of SNHG4 might be associated with progression of pneumonia.

Overexpression of SNHG4 attenuated LPS-induced in ammation and injury in cells
To explore the effect of SNHG4 in cell model of pneumonia in human lung broblast cells, WI-38 cells transfected with pcDNA-SNHG4 or pcDNA-3.1 were treated with 10 µg/mL of LPS. The results in Fig. 2A revealed that pcDNA-SNHG4 transfection could signi cantly increase SNHG4 expression in LPS-induced WI-38 cells. Furthermore, SNHG4 overexpression facilitated LPS-treated WI-38 cell proliferation and migration and restrained apoptosis (Fig. 2B-2E, P < 0.01). The levels of both IL-6 and TNF-α were increased in WI-38 cells under LPS treatment, which was reversed by SNHG4 upregulation (Fig. 2F, P < 0.01). Moreover, compared with LPS alone treatment group, the level of SOD was increased and MDA was decreased signi cantly after overexpression of SNHG4 in LPS-treated cells (Fig. 2G-2H, P < 0.01). These results suggested a protective role of SNHG4 overexpression in LPS-induced cell model of pneumonia in human lung broblast cells by reducing cell apoptosis and in ammation.

SNHG4 negatively regulated METTL3 expression in human lung broblast WI-38 cells.
To further investigate the underlying mechanism of SNHG4 in the regulation of pneumonia progression, we predicted the downstream targets of SNHG4 by online bioinformatic tool and found that SNHG4 might bind to the METTL3 promoter region. Interestingly, we found that the expression of METTL3 in pneumonia patient serum and LPS incubated cells was signi cantly higher than that in normal serum and cells (Fig. 3A and 3B, P < 0.01). We used Pearson correlation test to evaluate the relationship between SNHG4 and METTL3, and the ndings suggested that there was a strongly negative correction between SNHG4 and METTL3 levels (Fig. 3C, P < 0.01). Besides, RIP results showed that SNHG4 could combine with METTL3 in WI-38 cells (Fig. 3D, P < 0.01). The m 6 A quantitative analysis unveiled that the percentage of m 6 A content in the total RNA was markedly decreased in the SNHG4 overexpressed cells in the present of LPS treatment (Fig. 3D, P < 0.01). The mRNA level of METTL3 in WI-38 cells were memorably upregulated after LPS treatment, whereas SNHG4 overexpression could reversed this result (Fig. 3F, P < 0.01). Overall, these ndings concluded that SNHG4 inhibited METTL3 expression upregulation in in pneumonia patient serum and LPS-treated cells, which was also correlated with m 6 A content.

Overexpression of SNHG4 alleviated LPS-induced in ammatory damage by inhibiting METTL3 expression
To elucidate whether SNHG4 functions by regulating METTL3, rescue experiments were performed with co-transfection of pcDNA-SNHG4 with pcDNA-METTL3 plasmid. The e ciency of pcDNA-METTL3 on its expression level in WI-38 cells was veri ed by Western blotting (Fig. 4A and 4B). The results indicated that the pcDNA-METTL3 signi cantly restrained the proliferation, migration and SOD content, as well as promoted apoptosis, in ammatory factor contents and MDA concentration of WI-38 cells and reversed the effects on these processes induced by SNHG4 upregulation (Fig. 4C-4K, P < 0.05). Collectively, these data demonstrate that METTL3 exerts an injury promoting effect on WI-38 cells and serves a crucial function downstream of SNHG4.

Interference of METTL3 decreases the m 6 A level of STAT2 mRNA
As shown in Fig. 5A, with the help of online bioinformatics tools, we found that there were m 6 A binding sites on STAT2 mRNA. The m 6 A binding sequence in the STAT2 promoter region is 'GGACT'. The m 6 A quantitative analysis revealed that the m 6 A global methylation quantity is downregulated in the METTL3 inhibited WI-38 cells (Fig. 5B, P < 0.01). RIP-qPCR revealed that METTL3 silencing signi cantly reduced SOCS3 mRNA enrichment precipitated by the antibody (Fig. 5C, P < 0.01). The e ciency of METTL3 siRNA on its expression level in WI-38 cells was veri ed by Western blotting (Fig. 5D-5E, P < 0.01).
Moreover, METTL3 knockdown observably decreased SOCS3 mRNA and protein levels ( Fig. 5D and 5F-5G, P < 0.01). YTH m 6 A RNA-binding protein 1 (YTHDF1) is known to promote translation of m 6 A methylated transcripts [15]. The expression of STAT2 appeared to be promoted by m 6 A methylation, which raises the possibility that it is a target of YTHDF1. As expected, RIP-qPCR analysis revealed that STAT2 is a target gene of YTHDF1 (Fig. 5H). Overexpression of YTHDF1 can partially rescue the reduced STAT2 mRNA and protein expression levels caused by METTL3 knockdown in WI-38 cells (Fig. 5I-5J), con rming that YTHDF1 is involved in regulation of STAT2.

Overexpression of STAT2 reversed the ameliorative effect of SNHG4 upregulated on LPS-induced in ammatory damage
As STAT2 is a structural target that functions downstream of METTL3 and SNHG4, we further evaluated whether SNHG4 and STAT2 are functionally associated, pcDNA-SNHG4 and pcDNA-STAT2 was transfected alone or together into LPS-treated WI-38 cells. The e ciency of pcDNA-STAT2 on its expression level in WI-38 cells was veri ed by Western blotting (Fig. 6A and 6B). Subsequently, with CCK-8, Annexin V-FITC/PI, Transwell and ELISA assays, STAT2 overexpression was demonstrated to inhibit the proliferation, migration and SOD content, as well as facilitate the apoptosis, in ammatory factor concentration and MDA content of WI-38 cells. Further, we observed that the inhibitory effects on malignant biological behaviors and in ammatory factors secretion after SNHG4 overexpression could be largely blocked by STAT2 overexpression (Fig. 6C-6K). Collectively, our data suggest that the inhibitory role of SNHG4 in maintaining LPS induced injury was largely dependent on the METTL3/STAT2 axis.

Overexpression of SNHG4 alleviates LPS-induced in ammation and injury in mice
To further investigate, we intratracheally injected SNHG4 overexpression vector into mice. Consistent with in vitro ndings, compared with the control group, LPS treatment prominently inhibited SNHG4 expression and promoted the protein levels of METTL3 and STAT2, which were observably reversed by SNHG4 upregulation (Fig. 7A-7D, P < 0.01). As well, compared with the LPS treated group, the overexpression of SNHG4 inhibited in ammatory factor expression and MDA concentration, and restored SOD content ( Fig. 7E-7H, P < 0.05). In addition, LPS stimulation increased wet dry mass ratio (W/D) and myeloperoxidase (MPO) contents of isolated lungs, while pcDNA-SNHG4 injection decreased W/D and MPO, indicating that SNHG4 could suppressed pulmonary edema (Fig. 7I-7J, P < 0.05). Then, we collected and counted the macrophages and neutrophils in the alveolar lavage uid, and LPS injection promoted the number of macrophages and neutrophils, while SNHG4 overexpression inhibited the aggregation of macrophages and neutrophils (Fig. 7K-7L, P < 0.01).

Discussion
NP morbidity and mortality rate are very high, seriously impact on the physical health of newborn [4]. In this study, LPS was used for inducing WI-38 cells to establish a pneumonia in ammatory damage model. First, we found that LPS meaningfully inhibited cell viability and migration, enhanced apoptosis, and increased the levels of in ammatory factors IL-6 and TNF-α and oxidative stress. This is consistent with previous ndings. Zhang et al. Found that LPS induced WI-38 injury model could inhibit cell viability and enhance cell apoptosis at the cellular level, and increase the expression levels of Bax and cleavedcaspase-3 and the contents of IL-6 and MCP-1 at the molecular level [16]. Further studies revealed that LPS negatively regulated SNHG4 expression. Several lncRNAs have been proposed to regulate LPS induced cell models in pneumonia. For instance, a research reported that lncRNA XIST was highly expressed in patients with acute stage of pneumonia. Knockdown of XIST remarkably alleviated LPSinduced cell injury through increasing cell viability and inhibiting apoptosis and in ammatory cytokine levels through regulating JAK/STAT and NF-κB pathways [17]. lncRNA MIAT2 protected WI-38 cells from LPS injury and apoptosis through miR-15 / p38MAPK crosstalk, and ultimately affected the development of pneumonia [6]. Moreover, another study found that SNHG4 could regulate STAT2 and repress in ammation by adsorbing miR-449c-5p in microglia during cerebral ischemia-reperfusion injury [7]. Here, we observed that SNHG4 expression in serum of patients with pneumonia was signi cantly downregulated, and pcDNA-SNHG4 pre-transfection prominently inhibited the negative effects of LPS treatment on WI-38 cells and mouse lung tissue, suggesting that SNHG4 may participate in the anti LPS induced in ammatory injury in vitro and in vivo. In addition, we found that SNHG4 could bind and negatively regulate the expression of METTL3.
Recently accumulating data showed that METTL3, a methylation regulator of m 6 A, was dysregulated in various types of tumor and in ammatory diseases by affecting cell proliferation, invasion and apoptosis.
For example, LPS could enhance the expression and biological activity of METTL3 in macrophages, while overexpression of METTL3 signi cantly attenuated the in ammatory response induced by LPS in macrophages [18]. Overexpression of METTL3 promoted the activation of the TRAF6-NF-κB pathway in an m 6 A-dependent manner, further inhibiting NF-κB attenuated METTL3-mediated microglial activation and promoting LPS-induced microglial in ammation [12]. Notably, previous studies mainly focused on the effects of METTL3 on mRNA stability and translation, and little is known about whether it is regulated by lncRNAs to participate in disease initiation and progression. We found that LPS could enhance METTL3 expression at the mRNA levels in a dose-dependent manner in WI-38 cells. Besides, SNHG4 inhibited METTL3 expression upregulation in in NP patient serum and LPS-treated cells, which was also correlated with m 6 A content. Speci cally, in WI-38 cells, SNHG4 upregulation was accompanied by decreased mRNA stability and protein expression of METTL3 and its associated m 6 A methylation levels, which resulted in promotion of cell proliferation as well as inhibition of apoptosis. A previous study revealed that LNC942 speci cally binds to the METTL14 protein and a speci c motif 'GCAGGG' within the sequence. Thereafter, METTL14 regulated by LNC942 promotes the stability and expression of m 6 A methylation levels and its target genes, such as CXCR4 and CYP1B1 [19]. In this study, the promotion was almost restored by exposure to overexpression of SNHG4 and METTL3 in vitro, followed by reversal of the expression patterns of the above genes.
Janus kinase (JAK) / signal transducer and activator of transcription (STAT) pathway plays an important role in cytokine mediated biological response and is a classic in ammation related pathway. STAT may contribute to the development of acute lung injury in ammation. A research revealed that Azd1480, STAT2 inhibitor, could signi cantly reduce lung injury, reduce protein leakage and inhibit in ammatory cytokines release [20]. Cooperative networks of posttranscriptional modi cation pathways may ultimately regulate cell fate determination or stress by coordinating mRNA stability, translation e ciency, and splicing of transcripts that maintain cell type speci c proteomes. m 6 A modi cation is a dynamic and reversible process mediated by three m 6 A key elements ('Writers', 'Erasers' and 'Readers'). m 6 A reader proteins are also necessary in this process, including YTHDF1.The current study showed that METTL3 was responsible for catalytically installed m 6 A, and YTHDF1 were identi ed as ' readers' of m 6 A to regulate the stability of m 6 A bearing transcripts. YTHDF1 can destabilize m 6 A-containing mRNA to control the expression of key genes in multiple biological processes [21]. A study has found that METTL3 recruited YTHDF1 to enhance HK2 stability, thus promoting the Warburg effect in cervical cancer, which may facilitate new insights into cervical cancer treatment [22]. In this study, we found that m 6 A modi cation regulated STAT2 expression in a YTHDF1 orchestrated manner. Mechanistically, YTHDF1 recognizes and binds m 6 A-containing mRNA of STAT2, promotes translation and protein expression. Similarly, a recent study demonstrated that silencing METTL3 signi cantly promotes adipogenesis in porcine BMSCs by targeting the JAK1/STAT5/C/EBPβ pathway through an m 6 A-YTHDF1-dependent regulatory mode [23]. Further, we found that STAT2 was modi ed by m 6 A, and RIP-qPCR con rmed that STAT2 was a target of YTHDF1. Overexpression of YTHDF1 can partially rescue the reduced STAT2 mRNA and protein expression levels caused by METTL3 knockdown in WI-38 cells. A previous study has shown that METTL3 promoted STAT3 protein expression by regulating the translation of m 6 a-YTHDF1 dependent pathway [24], which further con rmed our conclusion.
In summary, we found that SNHG4 was downregulated in the serum of patients with NP and its overexpression could inhibit LPS induced in ammatory injury in human lung broblasts and mouse lung tissue. The molecular mechanism underlying this protective effect was achieved by suppression of METTL3-mediated m 6 A modi cation levels of YTHDF1-dependent STAT2 mRNA. These ndings contribute to a more extensive and in-depth understanding of the mechanism of lncRNAs in the occurrence and development of NP, and lay the foundation for nding a new target for the treatment of NP.

Funding
Not applicable

Competing interests
The authors declare that they have no competing interests.

Availability of data and materials
The datasets used during the present study are available from the corresponding author on reasonable request.     The contents of IL-6, TNF-α, SOD and MDA was detected by ELISA in WI-38 cells. Data were presented as mean ± SEM. N=5, **P < 0.01.

Figure 5
Interference of METTL3 decreases the m6A level of STAT2 mRNA A: Online bioinformatics tools (http://www.cuilab.cn/sramp) revealed the distribution of m6A peak on STAT2 mRNA. B: m6A quantitative analysis illustrated the m6A level with or without METTL3 silencing. C: RIP-qPCR (RNA immunoprecipitation following qPCR) showed the STAT2 mRNA enrichment precipitated by m6A antibody. D-E and G: Western blotting was used to illustrate the METTL3 and STAT2 protein expression with LPS administration and METTL3 silencing transfection. F: RT-qPCR was used to illustrate the STAT2 mRNA expression with LPS administration and METTL3 silencing transfection. H: RIP analysis of the interaction of STAT2 with YTHDF1 in WI-38 cells transfected with pcDNA-YTHDF1 plasmid. Enrichment of STAT2 with YTHDF1 was measured by qPCR and normalized to input. I: RT-qPCR of STAT2 mRNA in WI-38 cells with or without METTL3 knockdown and transfected with control or pcDNA-YTHDF1 plasmid.
J: Western blotting of STAT2 protein in WI-38 cells with or without METTL3 knockdown and transfected with control or pcDNA-YTHDF1 plasmid. Data were presented as mean ± SEM. N=5, **P < 0.01.