N6-methyladenosine-mediated Nrf2 Regulates the Defense Mechanism Against PM2.5-induced Pulmonary Fibrosis

Background: It has been reported that particulate matter with an aerodynamic diameter of < 2.5 µm (PM2.5) could induce epithelial–mesenchymal transition (EMT)- and extracellular matrix (ECM)-related pulmonary brosis (PF). The transcription factor Nrf2 alleviated PM2.5-induced PF by antagonizing oxidative stress. The N 6 -methyladenosine (m 6 A) modications play a signicant role in the stress response. However, the effect of m 6 A modication on the mechanisms of Nrf2-mediated defense against PM2.5-induced PF remain unknown. Here, we investigated the role and the underlying molecular mechanisms of m 6 A methylation of Nrf2 mRNA in PM2.5-induced PF. Results: Male C57BL/6 mice were exposed to ltered air (FA), unltered air (UA) and concentrated air (CA)for 16 weeks. 16HBE cells were treated with 0, 50, or 100 µg/mL PM2.5 for 24 h. Our data showed that chronic PM2.5 exposure could induce brosis in lung and increase Nrf2 signals. In Nrf2 decient cells, α-SMA expression was signicantly upregulated whereas E-cadherin decreased compared with WT cells after PM2.5 treatment which implied the aggravated brosis. m 6 A methyltransferase METTL3 was upregulated after PM2.5 treatment. m 6 A-methylated RNA immunoprecipitation (MeRIP) and qRT-PCR results showed that METTL3 improved the m 6 A modication of Nrf2 mRNA in PM2.5-exposed 16HBE cells. MeRIP-Seq and single-base T3 ligase-based PCR results showed that the m 6 A-modied sites of Nrf2 mRNA were 1317, 1376, and 935 in lung of mice after PM2.5 exposure. RIP results suggested that the m 6 A binding proteins YTHDF1/IGF2BP1 promoted Nrf2 translation by binding to Nrf2 mRNA m 6 A residues. Conclusions: Our results revealed the mechanism by which

Environmental pollutants can alter global N 6 -methyladenosine (m 6 A) levels and the expression of RNA methylation modulator genes, thereby inducing injury [13]. Our previous study found that m 6 A-regulated miRNA-126 processing activated the PI3K-AKT-mTOR pathway and exacerbated PF in rats after nanoscale carbon black particle inhalation [14]. It has been reported that m 6 A-regulated miR-873-5p can mediate the Keap1-Nrf2 pathway to resist colistin-induced oxidative stress [15]. Furthermore, m 6 A-mediated nuclear receptor PPaRα mRNA stability to regulate lipid metabolism during oxidative stress, which was induced by circadian clock change in mice liver [16]. In our previous study, m 6 A was concentrated in 3′-untranslated region (UTR) in Nis-treated 16HBE cells [17]. Zhao et al. found Nrf2 mRNA m 6 A methylation in prepubertal testicular tissue of rats exposed to di-(2-ethylhexyl) phthalate (DEHP) [18], suggesting the presence of potential m 6 A methylation sites in the 3′-UTR of Nrf2 mRNA. However, the m 6 A methylation levels and sites of Nrf2 mRNA, as well as its regulation mechanism on PM2.5-induced PF, is still unknown.
In this study, we established PM2.5 exposure models in mice and 16HBE cells. Our data identi ed that altered m 6 A modi cation of Nrf2 mRNA could affect the development of PF induced by PM2.5. Furthermore, we determined the exact Nrf2 mRNA m 6 A sites (1317, 1376, and 935) which enhanced Nrf2 translation by binding the m 6 A binding proteins, such as YTH domain-containing proteins (YTHDFs) and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs). The m 6 A-mediated Nrf2 signaling pathway might be used as a potential index to prevent PM2.5-induced PF.

PM2.5 collection and extraction
Airborne PM2.5 was collected on a Te on ® lter by a high-volume air sampler (Thermo Scienti c, Franklin, MA, USA) operating at a ventilation frequency of 18-20 h for 6 h per day, and the lter membranes were replaced every 3 days. The retrieved lter membranes were divided into small pieces, placed in a 50-mL centrifuge tube, ultrasonicated in iced water for 30 min, and subsequently shaken for 20 min three times each. The resulting PM2.5 extracts were passed along sterile 40-μm nylon lters (Corning Life Sciences, Corning, NY, USA), freeze-dried, packed in tinfoil, and preserved at −80 °C.

Animals and PM2.5 exposure
Six-week-old male C57BL/6 mice were obtained from Vital River Laboratory (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China). Standard food and water were provided with all the mice, which were housed under a 12 h light/dark cycle and standard conditions set to 50% humidity at 25-26 °C.
The mouse body weights were recorded. Twenty-four mice were randomly divided into three groups after 1 week of acclimatization. The mice were exposed to concentrated air (CA), un ltered air (UA), and ltered air (FA) PM2.5 according to our previously reported procedures [19]. The FA and UA mice were housed in the chambers with or without high e ciency particulate air lters, respectively [20]. CA mice were housed in the chamber with the concentrated PM2.5 by the PM2.5 concentration enrichment system (Beijing Huironghe Technology Co., Ltd, Beijing, China). All the mice were exposed for 6 h per day (from 9:00 to 15:00), 7 days per week for 16 weeks and housed in an individually ventilated cage (IVC) during the remaining time. The mice were then sacri ced, and their lungs were separated and weighed. All experimental protocols were approved in advance by the Committee of the Ethics Animal Experiments of Hebei Medical University (IACUC-Hebmu-20170163).

Cell culture and treatment
The human bronchial epithelial cell line (16HBE) was gifted by Dr. D. C. Gruenert (University of California, San Francisco, CA, USA). The cells were cultured in basic Dulbecco's Modi ed Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a water-saturated atmosphere under 5% CO 2 at 37 °C. All the mycoplasma contamination tests conducted using a polymerase chain reaction (PCR)-based universal mycoplasma detection kit were negative. The medium was replenished every day. A 0.05% solution of pancreatin ethylenediaminetetraacetic acid (EDTA; Gibco) was used to digest the cells when they completely covered the bottom of the culture dish.
The cells were transferred to 6-well plates with DMEM and starved for 12 h when the cells covered 80-90% of the board. The cells were then treated with 0, 50, or 100 μg/mL PM2.5 for 24 h.

Histopathological analysis and immunohistochemical (IHC) staining
Lungs were xed with 4% paraformaldehyde (3 mice per group). The para n-embedded lung tissues were cut into 5-μm continuous sections then stained with hematoxylin and eosin (H&E). According to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA), Masson's trichrome staining were performed to evaluate the distribution of brous collagen in lung.
IHC was used to detect the Nrf2, HMOX-1, METTL3 expression in lung tissue. The details were in additional les 1.
Determination of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) concentrations in mouse lung tissues by high-performance liquid chromatography (HPLC) The concentrations of MDA, SOD, and GSH in lung tissues of mice were determined using HPLC (Shimadzu Corporation, Kyoto, Japan). Lung tissues (0.5 mg) were homogenized on ice and then centrifuged for 15 minutes at 4 °C, 20,000 rpm, and the resulting supernatants were diluted with a mobile phase (including 1 mM EDTA) mixed at a 1:1 (v/v) ratio, centrifuged again for 2 min, and then instantly analyzed using HPLC.

Measurement of ROS concentrations
The intracellular ROS concentration in lung of mice was detected using 2,7-dichloro uorescein diacetate (DCFH-190 DA) labeling (Beyotime Biotechnology, Jiangsu, China) according to our previously study [21]. ROS levels were evaluated by the average uorescence intensity using ow cytometry (FACS) analysis (AccuriTMC6 ow cytometer, Becton Dickinson, Franklin Lakes, NJ, USA). The results were expressed as the mean ± standard deviation (SD). Six mice in each group were used to measure the ROS levels in lungs.

Western blot
The lysates of lung tissues or 16HBE cells were denatured by boiling for 10 min. The samples were loaded onto a polyacrylamide gel (PAG) containing 8-10% sodium dodecyl sulfate and were separated using

Stabilization of cell lines using lentiviral transduction
The stable cell lines were generated according to our previously described methods [22]. Brie y, LentiCRISPR V2 and Lenti PLEX-MCS vector were used for METTL3 knockdown or overexpression, respectively. Lentivirus vector, the packaging vector PAX2, and VSVG were cotransfected into 293T cells to prepare lentivirus. Cells were transduced using the forementioned lentiviruses with 8 μg /mL polybrene The MeRIP assay was performed according to a previously described protocol [23]. Brie y, RNA was extracted by TRIzol TM according to the manufacturer's instructions. The RNA was then fragmented using an RNA fragmentation kit (Ambion, Austin, TX, USA). Pierce M Protein A/G Magnetic Beads (Thermo Scienti c) were incubated with the m 6 A antibody (Synaptic Systems, Goettingen, Germany) at 4 °C for 3 h. The mixture of antibody-coupled magnetic beads, RIP buffer, and fragmented RNA was incubated at 4 °C overnight, and m 6 A nucleotide solution (Sigma-Aldrich) was used to elute the immunoprecipitated RNA for qRT-PCR analysis.
L-azidohomoalanine labeling of synthesized proteins The synthesized proteins were labeled with L-azidohomoalanine (AHA) according to a previously described method [24], with minor modi cations. Brie y, cells were incubated in a methionine-free medium for 30 min and then incubated in 50 µM AHA. The protein from cell lysates (500 µg) was used for click reactions according to Click-iT TM Protein Reaction Buffer Kit's protocols (Invitrogen). The total proteins obtained from the click reactions were precipitated with methanol and dissolved in buffer (50 mM Tris, 0.01% SDS). The biotin-labeled proteins were incubated with 50 µL streptavidin-conjugated magnetic beads (Dynabeads ® M-280 Streptavidin, Invitrogen) at room temperature for 5 h and washed with PBS and 0.5% SDS ve times. The immunoprecipitated proteins were analyzed using SDS-PAGE and western blotting with Nrf2 antibodies.

Single-base m 6 A validation using ligase-based PCR
A re ned single-base T3 ligase-based method was described previously [25]. Brie y, the designed DNA probes L and R matched the anking sequences of the m 6 A site (additional les 4). Probes L and R were modi ed with phosphate at the 5' terminal and two ribonucleotides at the 3′-end codon. The 20 nm probes L and R, 1 × T3 ligation buffer (New England Biological Laboratory [NEB], Ipswich, MA, USA), and 300 ng total RNA were mixed and incubated at 85 ° C for 3 min, then at 35 °C for 10 min. T3 DNA ligase (NEB) was added to the nal volume of 10 μL.
The mixture was incubated at room temperature for 10 min and then immediately chilled on ice. 1 µL ligation product was ampli ed by PCR for 24 cycles. The samples were loaded on 2-3% agarose gels and detected by electrophoresis.
The sequences of siRNA oligonucleotides are listed in additional les 5. 16HBE cells (5 × 10 5 /mL) were temporarily transfected with siRNA using Lipofectamine ® RNAiMAX transfection reagent (Invitrogen) based on the manufacturer's instructions. The knockdown effects were analyzed using western blotting.

RIP
The CDS regions of IGF2BP1 were synthesized and cloned into pcDNA3 FLAG 2AB vector by the HindIII and EcoRI restriction sites. The 2AB-FLAG-IGF2BP1 and 2AB-FLAG-YTHDF1 plasmids were transfected into 10 7 cells using Lipofectamine ® 3000 for 48 h. Then, the cell lysate was immunoprecipitated with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). Magnetic beads coated with 5 µg antibodies against IgG (Millipore) or FLAG ® (Sigma Aldrich) were incubated with prefrozen cell lysates overnight at 4°C . The magnetic bead-bound complexes were washed with RIP washing buffer ve times. The precipitated RNA was extracted by a puri ed phenol-chloroform solution and analyzed using qPCR. The cloning primers of 2AB-IGF2BP2-CDS and 2AB-IGF2BP3-CDS are shown in additional les 3.

Statistical analysis
All the results are expressed as the mean ± SD. Statistical analysis was performed using SPSS 24.0 software (SPSS ® ; IBM ® , USA). Unpaired two-tailed Student's t-tests were used to compare the differences between two groups. One-way analysis of variance (ANOVA) was used to compare the differences among three or more groups with Dunnett's or the least signi cant difference (LSD) post-hoc test. P < 0.05 represented a statistically signi cant difference.

PM2.5 concentration and physical characteristics
The mice were exposed to PM2.5 from December 1, 2017 to March 25, 2018. During the exposure period, the PM2.5 concentrations were an average of 0, 86.78, or 671.87 µg/m 3 in the FA, UA, or CA chambers, respectively. Our results showed that 91.22% and 99.49% of the particles in the CA and UA chambers were PM2.5, respectively (Table 1). Meanwhile, no particles were observed in the FA chamber. Besides the PM, no statistical signi cance was observed for the other air components, such as the SO 2 , CO, NO 2 , or O 3 in different chambers.

PM2.5 induced PF and collagen deposition in lung tissues of mice and 16HBE cells
In CA and UA mice, the histopathological results showed that the alveolar walls and bronchiole structure were disordered compared to FA mice (Fig. 1A). Meanwhile, the alveolar walls were thickened, the alveolar septa were narrowed, and the bronchial epithelial cells had proliferated in both CA and UA mice. Numerous in ammatory cells in ltrated into interstitium. Macrophages, monocytes, lymphocytes, and brous nodules were found in the lungs of the CA and UA mice. Speci cally, damaged alveolar structures, thickened diaphragms, broblast formation, and increased collagen bers were found in the CA and UA mice. The results of Masson's trichrome staining indicated positive staining representing metagenetic brosis in the lungs of mice after PM2.5 exposure. Compared with the FA mice, positive staining areas were signi cant increase in CA and UA mice (P < 0.01) (Fig. 1A).
Nrf2 defends against oxidative stress in lung and 16HBE cells after PM2.5 treatment Oxidative stress in lungs of mice ROS levels in the lungs of CA mice increased by 2.38-fold and 1.95-fold compared with FA and UA mice, respectively (P < 0.05) ( Fig. 2A). Our data showed that the MDA levels increased by 1.28-fold in CA mice (P < 0.05) but slightly increased in UA mice (P > 0.05) compared with the FA mice (Fig. 2B). Compared with the FA mice, the SOD levels decreased by 1.32-and 1.66-fold in UA and CA mice, respectively (P < 0.01) (Fig.  2C). The GSH concentration decreased by 1.35-fold in CA mice compared with the FA mice (P < 0.05) (Fig.  2D). All above data demonstrated elevated oxidative stress in lung of mice after PM2.5 exposure.
Changes of Nrf2-related proteins in lung of mice and 16HBE cells after PM2.5 exposure IHC staining was performed for Nrf2 and HMOX-1 expression in lung tissues. In the FA group, Nrf2 and HMOX-1 were expressed in the pulmonary vascular and airway smooth muscle cells, while no obvious positive staining was observed in other areas. After PM2.5 exposure, Nrf2 and HMOX-1 expressed in the broblast cytoplasm, except for above areas (Fig. 2E-G). As shown in Fig. 2H, the Nrf2 signal-related proteins expressed in mice lung tissues were detected by western blot. Nrf2 expression signi cantly increased by 1.15-fold and 1.8-fold in UA and CA groups compared with FA group, respectively (P < 0.05) (Fig. 2I). HMOX-1 expression signi cantly increased by 1.28-fold and 1.68-fold in UA or CA mice compared with the FA mice, respectively (P < 0.05) (Fig. 2J). No signi cant difference was observed in Cul3 and Keap-1 protein expression (P > 0.05) (Fig. 2K, L). Nrf2 expression increased signi cantly in 100 μg/mL PM2.5treated 16HBE cells compared with the control (P < 0.05) (Fig. 2M, N). HMOX-1 protein levels signi cantly increased in 100 μg/mL PM2.5-treated 16HBE cells compared with the control (P < 0.01) (Fig. 2O). Cul3 and Keap-1 expression showed no signi cant difference between groups (P > 0.05) (Fig. 2P, Q). The levels of METTL3 protein in lung of mice were detected by IHC staining (Fig. 3A, B). The results of qRT-PCR showed that METTL3 mRNA expression was signi cantly upregulated by 1.58-fold and 1.11-fold in CA and UA groups compared with FA group, respectively (P < 0.05) (Fig. 3C). Western blotting showed that the METTL3 expression increased by 1.55-fold and 1.09-fold in CA and UA groups compared with FA group, respectively (P < 0.05) (Fig. 3D, E). However, METTL14, ALKBH5, and FTO mRNA expression was not signi cantly different among the different groups (Fig. 3F-H). In 16HBE cells, western blotting suggested that METTL3 expression increased by 1.66-fold in 100 µg/mL PM2.5 group compared with 0 µg/mL PM2.5 group (Fig. 3I, J).

m 6 A modi cation mediated Nrf2 mRNA translation
The m 6 A modi cation data were extracted from the GEO datasets and m 6 A motifs within the Nrf2 CDS at a single-nucleotide resolution by analyzing the data from the m 6 A iCLIP (miCLIP)-Seq (additional les 6 Fig.   S3). Based on the sequence, we designed three pairs of primers to detect the m 6 A modi cation (Fig. 4A). m 6 A MeRIP-qRT-PCR con rmed that m 6 A-modi ed Nrf2 expression was signi cantly decreased in KO-M3 cells compared with V2 cells (Primer 2, Primer 3) (P < 0.01) (Fig. 4B). Furthermore, m 6 A-modi ed Nrf2 expression was signi cantly increased in 100 μg/mL PM2.5-treated 16HBE cells compared with 0 μg/mL PM2.5-treated 16HBE cells (Primer 2, Primer 3) (P < 0.01) (Fig. 4C). qPCR showed no signi cant differences of Nrf2 expression in 50 and 100 μg/mL PM2.5-treated 16HBE cells compared with 0 μg/mL PM2.5-treated 16HBE cells (P > 0.05) (Fig. 4D). Our data suggested that expression of Nrf2 protein (Fig. 2N), but not that of the corresponding mRNA, was upregulated in the PM2.5-exposed 16HBE cells. To support this result, we next monitored the expression of the Nrf2 synthesized in KO-M3 and V2 cells by incorporating CLICK chemistry and AHA. The V2 and KO-M3 cells were labeled with AHA, and the resulting AHA-labeled synthesized proteins were biotinylated and affinity-purified with streptavidin. Although the V2 cells robustly synthesized Nrf2 protein, protein synthesis was almost completely blocked in KO-M3 cells (Fig. 4E, F).

Single-base m 6 A detection identi ed exact Nrf2 mRNA m 6 A sites
It has previously been reported that Nrf2 has ve m 6 A sites (808, 935, 1317, 1333, and 1376). m 6 A motifs within the CDS of Nrf2 are shown from the date of MeRIP-Seq in FA and CA mice. The results showed that m 6 A enrichment increased by 1.41-fold in CA mice compared with FA mice (P < 0.01) (Fig. 4A, additional les 6 Fig. S3). To further determine the exact Nrf2 mRNA m 6 A sites related to PM2.5 exposure, we designed probes L and R targeting the Nrf2 CDS and used the T3 ligase to concatenate both probes on templates, which could be subsequently ampli ed by PCR. Furthermore, we designed probes against a nonmethylated Nrf2 A site as a control. Previous studies have suggested that the T3 ligase is highly selective between the m 6 A and A sites. Our results showed that the ligation e ciency was signi cantly decreased at the m 6 A sites compared with the nonmethylated A sites. Furthermore, the difference between the ligation e ciencies was magni ed by PCR. Therefore, the concentration of PCR products could be used to evaluate the ligation e ciency m 6 A and indicate the methylation level of each site [26]. Our results showed that only three sites (1317, 1376, and 935) were methylated in PM2.5-exposed 16HBE cells but not the other two sites (1333 and 808) (Fig. 4G). Furthermore, the methylation could promote Nrf2 mRNA translation after PM2.5 exposure (Fig. 4H).

Discussion
In this study, the exposures duration was 6 h/day for 16 weeks. Therefore, the average PM2.5 concentration during this period was equal to 21.67 μg/m 3 in UA chamber and 168 μg/m 3 in CA chamber for an average of 24 h average, which was lower than or similar to the levels in Shijiazhuang from December 1st, 2017 to March 25th, 2018, respectively. After short-term PM2.5 (271.6 ± 84.8 µg/m 3 for 1 day or 138.2 ± 34.3 µg/m 3 for 5 days) exposure, the broken alveolar walls enlarged spaces, and the interstitium lled with in ammatory exudates were observed in lungs of mice [27]. In our previous studies, carbon black nanoparticles (CBNPs), a major airborne particle, could induce alveolar macrophage accumulation and in ammatory cell in ltration and increase the incidence of PF in rat lung tissues [14,28]. Here, we observed thickened alveolar walls, narrowed alveolar septa, in ltrated in ammatory cells, and metagenetic brosis in lungs of UA and CA mice. Increased vimentin and decreased E-cadherin expression are hallmarks of EMT that usually contribute to brosis [5,29]. Collagen is also an extracellular component, and its abnormal accumulation is a key feature of PF [30]. Increased α-SMA expression whereas decreased E-cadherin were observed in lung of mice after PM2.5 treatment [31]. In the present study, vimentin and α-SMA expression increased whereas E-cadherin expression decreased in lungs and 16HBE cells after PM2.5 exposure. These results indicated the occurrence of brosis and increased EMT activity in lung of mice after PM2.5 exposure.
It has already been shown that PM2.5-induced oxidative stress is mainly caused by an imbalance between ROS production and antioxidant defense activity [32,33]. Excess ROS lead to oxidative stress, induce tissue damage and then promote brosis related with the antiapoptotic broblast formation in idiopathic pulmonary brosis (IPF) [34]. In the present study, signi cantly increased ROS levels were observed in lung of mice. Furthermore, PM2.5 exposure increased the MDA concentration in lung of mice. In contrast, we observed signi cant decreases of SOD activities and GSH levels after PM2.5 exposure. Our results indicated that the PM2.5 could increase the concentrations of superoxide radicals, hydrogen peroxide, and MDA, whereas downregulate the SOD activity and decrease the GSH levels to aggravate oxidative stress [35].
Nrf2 could alleviate oxidative stress, increase cellular proliferation and migration, and decrease apoptosis, which may modulate MMP9, TGFβ1, and bronectin expression [36]. Nrf2 is the primary transcription factor regulating HMOX1 mRNA expression [37]. In bleomycin-treated PF mice, Nrf2 expression increased after exposure for 7 or 14 days and decreased after 28 days [5,38]. In previous studies, increased Nrf2 protein was found in the lungs of mice [36] and prefrontal cortex of rats [39] after PM2.5 exposure. In the present study, Nrf2 expression was increased in the lungs of the mice exposed to PM2.5 for 8 or 16 weeks, possibly because continuous PM2.5 exposure might induce persistent oxidative stress and then increase Nrf2 expression (additional les 6 Fig. S6 for 8-week mice experiment). HMOX-1 is an enzyme showing strong anti-in ammatory and antioxidative properties. It catalyzes the degradation of the proin ammatory free heme and produces anti-in ammatory compounds, such as bilirubin and carbon monoxide, which was selected as a marker for inducing the cellular protective mechanism [40]. In a previous study, both of HMOX-1 expression and Nrf2 translocation increased in a cell alveolar model treated with diesel exhaust particulate matter [41]. In a traumatic brain injury (TBI) mouse model, Dong et al. found that both of Nrf2 mRNA and protein expression increased. Moreover, downstream HMOX-1 was upregulated at the transcription and translation levels. In Nrf2-KO C57BL/6 mice, Nrf2 deletion weakened HMOX-1 expression [42]. Zhang et al. also found more severe PF and lower HMOX-1 and NQO1 expression in bleomycin-treated Nrf2 -/mice compared with WT mice [5]. Oh et al. found that sulforaphane promoted the expression of Nrf2 and its downstream HMOX-1, which attenuated hepatic brosis [43]. Furthermore, Nrf2 expression in prefrontal cortexes of rats increased signi cantly after PM2.5 exposure for 12 weeks [39]. In our study, Nrf2 and HMOX-1 expression increased in mice and 16HBE cells after PM2.5 exposure. However, the Nrf2-related proteins Cul3 and Keap-1 were no signi cant differences in lung of mice and 16HBE cells after PM2.5 treatment. These ndings suggest that Nrf2, but not Cul3 or Keap-1, plays a major role in the PM2.5-induced PF model. Similarly, we found that HOMX-1 expression was regulated by Nrf2. Furthermore, increased vimentin levels were observed in lungs of mice and 16HBE after PM2.5 treatment. After Nrf2 deletion, vimentin expression increased whereas HMOX-1 expression decreased compared with the WT cells after PM2.5 exposure. Therefore, we demonstrated that Nrf2 could promote the antioxidative enzyme HMOX-1 and inhibit vimentin expression induced by PM2.5. α-SMA expression was signi cantly upregulated whereas E-cadherin levels were decreased in 16HBE siRNA-Nrf2 cells after PM2.5 treatment compared with that of 16HBE siRNA control cells. These data suggested that Nrf2 de ciency could aggravate PM2.5-induced PF by decreasing HMOX-1 and E-cadherin expression as well as increasing α-SMA and vimentin expression. m 6 A could regulate RNA metabolism, including pre-mRNA splicing, mRNA nuclear export, mRNA stability, mRNA translation [44]. In a previous study, METTL3 and METTL14 expression was observed increases in mice after PM2.5 exposure (271.6 ± 84.8 µg/m 3 ) but not found signi cant changes of FTO and ALKBH5 expression [27]. Similarly, in the present study, the levels of METTL3 transcription and protein were increased in lungs of mice and 16HBE cells after PM2.5 exposure but not about METTL14, ALKBH5 or FTO expression. Additionally, Wang et al. found that METTL3 might regulate Keap-1/Nrf2 pathway in colistininduced oxidative stress by interacting with DGCR8 [15]. In our study, Nrf2, and HMOX-1 expression was signi cantly decreased in KO-M3 cells compared with V2 cells after PM2.5 exposure. In KO-M3 cells, α-SMA levels were signi cantly upregulated compared with in V2 cells after PM2.5 treatment. Rescued METTL3 expression could increase the Nrf2 expression and decrease α-SMA levels. These data indicated that METTL3 suppression might impair the ability of the Nrf2 pathway to defend against PM2.5-induced oxidative stress and increase brosis.
In the present study, we found the expression of Nrf2 protein but not mRNA was upregulated in the PM2.5exposed 16HBE cells, which indicated that the m 6 A modi cation selectively mediated Nrf2 mRNA translation, but not its transcription. Our results indicated PM2.5-induced METTL3 expression promoted YTHDF1/IGF2BP1 to bind on Nrf2 mRNA through m 6 A modi cation and then induced Nrf2 translation.