Mechanism Of RNA Methylation Modification On The Pulmonary Fibrosis Induced By PM_2.5

Persistent air pollution threatens to human health.Respiratory diseases caused by air pollutants account for 47%of premature deaths worldwide each year.Exposure to Particulate Matters with Aerodynamic Diameter≤2.5μm(PM_2.5)could induce or aggravate respiratory diseases such as pulmonary dysfunction and fibrosis in patients with chronic lung diseases.Epigenetic inheritance affects gene selective expression and post-transcriptional regulation,which could explain the adverse health effects of exposure to environmental particulate matter to lung injury through the interaction between genes and environment.Non-coding RNA regulation is a form of epigenetic regulation that involves in the development of diseases.It has been found that the targeted regulation of mi RNA-m RNA plays a key role in the development of pulmonary fibrosis.Thus,differentially expressed mi RNAs is of great significance to further study the regulatory mechanism of PM_2.5-induced pulmonary fibrosis.N6-methyladenosine（m6A）is the most common RNA modification in eukaryotic m RNAs.m6A modification is jointly regulated by methyltransferase,demethylases and methylation modification recognition protein.Enzymes involved in m6A modification affect the differential expression of RNA,which regulated RNA splicing,processing,transcription,translation,degradation and affected the development of lung diseases.However,it remains unclear the regulatory mechanism of m6A modification-related enzyme-mediated RNA differential expression in PM_2.5-induced pulmonary fibrosis.m6A modification at different sites is closely related to biological functions.Therefore,accurate identification of m6A modification sites can provide targeted treatment strategies for PM_2.5-induced pulmonary fibrosis.It provides a scientific basis for the prevention and control of respiratory diseases caused by air pollutants.Part One Regulatory mechanism of EMT in pulmonary fibrosis induced by PM_2.5Objective:To study the epigenetic regulatory mechanism of Epithelial-Mesenchymal Transition（EMT）in pulmonary fibrosis induced by PM_2.5.Methods:1.Animal treatment and environment monitoring:Thirty mice were divided randomly into 3 groups and exposed to the filtered air（FA）,unfiltered air（UA）and the concentrated air（CA）,respectively.The FA mice were exposed to filtered air in identical chambers with a high-efficient particulate air filter.The UA mice were exposed to ambient air.The CA mice were exposed to concentrated PM_2.5.The total duration of exposure was performed 6h per day for 8 weeks.The real-time concentration of PM in chambers and ambient air were monitored by Aerosol Detector,and analyzed the distribution of the particulate matters using Aerodynamic Particle Sizer Spectrometer.The temperature and humidity in each chamber were measured continuously.2.After the end of exposure,the histopathological changes,fibrosis of lung tissues and EMT were detected by HE staining,Masson staining and immunofluorescence,respectively.3.A mi RNA microarray was used to profile the mi RNAs in serum.The expression of mi RNAs in lung tissues and serum were detected by q RT-PCR.Results:1.During exposure,the average PM_2.5 concentration was 0,94.84 and900.21μg/m³ in FA,UA and CA chambers,respectively.Particulate matter was not found in FA chambers.Diameter measurement showed that more than 91%particles in UA chambers were identified to be<2.5μm,and more than 99%particles in CA chambers were less than 2.5μm.The average temperature was21±2℃.And the average humidity was 35%-60%.2.Compared with the FA group,apparent lung histopathological changes and collagen deposition were observed in UA and CA mice.Immunofluorescence staining analysis showed that the expression of E-cad was decreased whereas the expression of Vimentin was increased in a dose-dependent manner in lung tissues of mice after PM_2.5 exposure for 8 weeks（P<0.05）.3.Microarray analysis showed that mice exposed to concentrated PM_2.5 for8 weeks induced the differential expression of 38 mi RNAs in serum（P<0.05）.Compared with the FA group,13 differentially expressed mi RNAs in the CA group were related to pulmonary fibrosis（P<0.05）.The 13 mi RNAs were mi R-3971,mi R-411-3p,let-7a-1-3p,mi R-93-3p,mi R-20b-3p,mi R-8118,mi R-615-5p,mi R-7663-3p,mi R-27a-5p,mi R-7013-5p,mi R-6996-5p,mi R-344g-5p,mi R-466m-3p.The targets of 13 mi RNAs were CRK,NR2F2,ACVR1,VIM,RASSF1,CCND2,PRKCA,SIRT1,CDK6,MAP3K7,HIF1A,UBE2V2,ATG10,BAX,RASSF1,E2F1,E2F4,SMAD5,TGFBR2,RASSF5,CTNNB1,BMP2,which was involved in the development of pulmonary fibrosis.Summary:In the early stage of PM_2.5 exposure,serum mi RNA plays an important role in the process of EMT-mediated pulmonary fibrosisPart Two Mechanism of YTHDF2 regulates EMT RNA methylation modification in PM_2.5-induced Pulmonary FibrosisObjective:To explore the regulatory mechanism of mi RNA-targeted YTHDF2 on RNA methylation in PM_2.5-induced pulmonary fibrosis.Methods:1.Animal treatment:Thirty mice were divided randomly into 3 groups and exposed to the FA,UA and CA,respectively.The total duration of exposure was performed for 16 weeks.2.Construction of cells models:During the exposure period,the ambient PM_2.5 was collected on the Teflon filter by High-Volume Air samples.The PM_2.5suspension was extracted by an ultrasonic processing in deionized water,and then dried in a vacuum frozen desiccator.BEAS-2B cells were treated with 0,25,50,and 100μg/ml of PM_2.5 extracts for 24h.3.The expression ofα-SMA、Collagen I、E-cad、Vimentin、N-cad、Mettl3、YTHDF2 protein were detected by Western blot.The Mettl3、Mettl14、FTO and CDH1 m RNA levels were elevated by q RT-PCR.4.For methylated RNA immunoprecipitation,the methylated RNA immunoprecipitation（Me RIP）assay.To detect the binding between CDH1m RNA and YTHDF2 protein,RNA-binding protein immunoprecipitation（RIP）assay was performed.For detection of CDH1 m RNA stability,the total RNA was isolated for q RT-PCR.5.According to the website database,the binding of YTHDF2 and mi R-494-3p was predicted.6.Cell transfection:For Mettl3 and YTHDF2 knockdown,BEAS-2B cells were transfected with si Mettl3,si Control,si YTHDF2 and si NC.The mi R-494-3p mimic,mimic NC,mi R-494-3p inhibitor and inhibitor NC were synthesized to upregulate or downregulate the expression of mi R-494-3p in BEAS-2B cells.7.In si Mettl3,si YTHDF2 and OE YTHDF2 cells,the effect of Mettl3 and YTHDF2 on pulmonary fibrosis were detected by Western blot.8.In mi R-494-3p mimic and mi R-494-3p inhibitor cells,the expression of YTHDF2 was detected by q RT-PCR and Western blot.9.To test the potential binding sites of YTHDF2 m RNA and mi R-494-3p,the dual luciferase reporter assay was performed.The rescue experiments were further detected the regulatory relationship between mi R-494-3p,YTHDF2 and CDH1 m RNA.Results:1.During the 16-week exposure period,average PM_2.5 concentration was0,86.78 and 671.87μg/m³ in FA,UA and CA chambers,respectively.2.Compared with FA group,the levels of a-SMA and collagen I protein were slightly increased in UA and CA group（P<0.05）.Compared with FA group,the levels of E-cad protein were decreased（P<0.05）,whereas the Vimentin and N-cad levels were increased in in UA and CA group（P<0.05）.In BEAS-2B cells,PM_2.5 treatment enhanced the protein expression ofα-SMA,collagen I,Vimentin,and N-cad（P<0.05）,whereas decreased the E-cad protein levels（P<0.05）.3.The levels of Mettl3 were increased in the lung of mice after PM_2.5exposure for 16 weeks（P<0.05）but the Mettl14 and FTO levels were no significant differences（P>0.05）.The levels of Mettl3 protein were slightly increased in BEAS-2B cells after PM_2.5 treatment in comparison to control group.The expression of E-cad protein in si Mettl3 cells was 1.9-fold increase compared with si Control cells after PM_2.5 treatment（P<0.05）.The expression of Vimentin and N-cad in si Mettl3 cells significantly decreased by 11.6%,28.8%in comparison to si Control cells after PM_2.5 treatment（P<0.05）,respectively.In me RIP assay,Mettl3 depletion significantly decreased m6A modification of the CDH1 m RNA with PM_2.5 treatment（P<0.05）.Compared with si Control cells,knockdown of Mettl3 had less effect on the transcriptional inhibition of CDH1m RNA.According to RNA stability assay,knockdown of Mettl3 had less effect on the transcriptional inhibition of CDH1 m RNA compared with si Control cells（P<0.05）.4.The expression of YTHDF2 protein was increased in the lung of mice after PM_2.5 exposure for 16 weeks and in PM_2.5-treated BEAS-2B cells.（P<0.05）RIP assay showed that CDH1 m RNA was pulled down by YTHDF2 antibody（P<0.05）.CDH1 m RNA exhibited significant transcriptional inhibition at different time points in si YTHDF2 cells after Act-D treatment（P<0.05）,compared with si NC cells.E-cad protein levels in si YTHDF2 cells were observed 2.42-fold increase compared with si NC cells after PM_2.5 treatment（P<0.05）.Compared with the si NC cells following by PM_2.5 treatment,the levels of Vimentin,N-cad,a-SMA,and collagen I relatively decreased by 14%,10%,36%,31%in si YTHDF2 cells（P<0.05）,respectively.E-cad protein expression was decreased（P<0.05）whereas levels of Vimentin,N-cad,a-SMA,and collagen I protein were increased in OE YTHDF2 cells（P<0.05）after PM_2.5treatment compared with OE NC cells.5.According to analysis of Targetscan,mi RDB,and Starbase databases,mi R-494-3p was predicted potentially to bind the same sequence in 3’UTR region of YTHDF2 m RNA in mouse and in human.In lung tissues of mice,mi R-494-3p was downregulated whereas YTHDF2 m RNA was upregulated in a dose-dependent fashion（P<0.05）.The downregulated mi R-494-3p expression and upregulated YTHDF2 m RNA expression were observed in BEAS-2B cells after PM_2.5 treatment（P<0.05）.The dual-luciferase reporter assays confirmed that YTHDF2 m RNA was targeted by mi R-494-3p（P<0.05）.6.The levels of YTHDF2 protein and m RNA in mi R-494-3p mimic cells had a reduction by 23.4%,47%in comparison to mimic NC cells after PM_2.5treatment（P<0.05）,respectively.Compared with inhibitor NC,the mi R-494-3p inhibitor significantly upregulated the levels of YTHDF2 protein and m RNA with or without PM_2.5 treatment（P<0.05）.The levels of CDH1 m RNA in mi R-494-3p mimic cells were 2.25-fold increase in comparison to mimic NC cells after PM_2.5 treatment（P<0.05）.Compared with inhibitor NC,the mi R-494-3p inhibitor significantly downregulated the expression of CDH1 m RNA with or without PM_2.5 treatment（P<0.05）.Summary:After PM_2.5 exposure,mi R-494-3p targets YTHDF2 to recognize m6A-modified CDH1 m RNA,promoting EMT processes and pulmonary fibrosis.Part Three Mechanism of ALKBH5 regulating site-specific RNA methylation in PM_2.5-induced pulmonary fibrosisObjective:To explore the potential mechanism of ALKBH5 regulating Atg13 m RNA methylation in PM_2.5-induced pulmonary fibrosis.Methods:1.Animal treatment:Twenty WT and Twenty KO male mice were randomly divided into FA and CA groups in this experiment,respectively.WT-FA and KO-FA mice received filtered air in the chambers.Meanwhile,WT-CA and KO-CA mice were exposed to concentrated PM_2.5.All mice were lasted for8-week exposure.2.Construction of cells models was the same as the Part Two.3.Autophagy in lung tissues was assessed using TEM.The immunofluorescence assays were used to detect the co-localization of LC3 and IκB-α.4.The expression of NLRP3、Caspase 1、Pro-caspase 1、IL-1β、ASC、NF-κB、IκB-α、LC3、SQSTM1、Atg13、Atg101、ULK1、FIP200、Mettl14、ALKBH5 protein were detected by Western blot.The Atg13、ULK1、RB1CC1m RNA levels were elevated by q RT-PCR.5.Cell transfection:For Atg13 knockdown,BEAS-2B cells were transfected with si Atg13 and si NC.6.The potential m6A-methylation sites were analyzed and retrieved from the website database.Determination of m6A at targeted sites was based on the single-based T3 ligase-based method.7.RCas9-ALKBH5 targeting site 767 of Atg13 m RNA was established to assess the effect of site-specific m6A modification on autophagy and inflammation in BEAS-2B cells.8.WT and ALKBH5^-/-mice were exposed to PM_2.5 for 8 weeks to further evaluate the role of ALKBH5 in regulation of autophagy,inflammation and fibrosis.The histopathological changes and fibrosis of lung tissues were detected by HE staining and Masson staining.Results:1.After PM_2.5.exposure,compared to FA group,the expression of NLRP3、Pro-caspase 1、Caspase 1、IL-1β、ASC、α-SMA and Collagen I protein in UA group and CA group.2.TEM imaging indicated that the autophagosomes and autolysosomes were increased in lung epithelial cells of mice exposed to PM_2.5 for 8 weeks and16 weeks（P<0.05）.In comparison with FA mice,the LC3 and SQSTM1 protein expression were dramatically increased in UA and CA mice exposed to PM_2.5for 8 weeks or 16 weeks,respectively（P<0.05）.The LC3 and SQSTM1 protein expression showed dose-dependent increase in BEAS-2B cells with PM_2.5treatment（P<0.05）.The induction of LC3 protein resulted from PM_2.5 exposure was relatively reduced by 3MA pretreatment or enhanced by Baf A pretreatment,respectively（P<0.05）.Double-immunofluorescence labeling indicated that colocalization of LC3 and IκB-αprotein in lung tissues of mice after 8 and 16weeks of PM_2.5 exposure.Significant increases of NF-κB protein in lung tissues were detected in UA and CA mice in comparison with FA mice（P<0.05）.Whereas,the IκB-αexpression was reduced in UA and CA mice in comparison with FA mice after 8-and 16-week of PM_2.5 exposure（P<0.05）.3.Compared to FA mice after PM_2.5 exposure,the FIP200,ULK1 and Atg13 protein expression showed significant increase in UA and CA mice（P<0.05）.The expression of FIP200,ULK1 and Atg13 protein was markedly increased in BEAS-2B cells in a dose-dependent manner（P<0.05）.Compared to NC cells,the expression of FIP200,ULK1 and LC3 was relatively decreased by 35.2%,48.3%and 34.5%in si Atg13 cells treated with PM_2.5（P<0.05）.Compared to PM_2.5-treated NC cells,the NF-κB,NLRP3,Pro-caspase 1,Caspase 1,IL-1βand ASC protein in si Atg13 cells had a reduction by 30.3%,63.6%,35.7%,52.4%,40.4%and 48.1%,relatively（P<0.05）.4.The expression of ALKBH5 protein in UA and CA mice exhibited a dose-dependent decrease in comparison with FA mice（P<0.05）.The Mettl3 and Mettl14 protein expression in UA mice and CA mice was markedly increased in comparison with FA mice after PM_2.5 exposure（P<0.05）.PM_2.5 treatment reduced ALKBH5 expression in BEAS-2B cells whereas induced Mettl3 and Mettl14 expression in a dose-dependent manner（P<0.05）.In comparison with FA group,ULK1 and Atg13 m RNA were upregulated in UA and CA group exposed to PM_2.5（P<0.05）.Significant upregulation of ULK1 and Atg13 m RNA（P<0.05）were observed in PM_2.5-treated BEAS-2B cells.No significant diffidence of RB1CC1 m RNA was observed neither in vivo nor in vitro following PM_2.5 exposure（P>0.05）.The expression of Atg13 m RNA was relatively decreased by 33.9%in ALKBH5 OE cells in comparison with OE NC cells treated with PM_2.5（P<0.05）.After PM_2.5 treatment,relative m6A enrichment（IP/Input）in ALKBH5 OE cells was decreased by 64.9%in comparison with OE NC cells（P<0.05）.5.Six potential m6A-methylation sites were analyzed and retrieved in website database.The site 767 of Atg13 m RNA expression in ALKBH5 OE cells were 1.8-fold higher than that in OE NC cells after PM_2.5 exposure（P<0.05）.The FIP200,ULK1 and LC3 protein expression in RCas9-ALKBH5targeting group had a relative reduction by 48.8%,33.2%and 35.6%in comparison with non-targeting control group after PM_2.5 treatment（P<0.05）.Compared to non-targeting control group,NF-κB,NLRP3,Pro-caspase 1,Caspase 1,IL-1βand ASC protein relatively reduced by 25.8%,39.1%,53.6%,32.5%,37.5%and 25.9%in RCas9-ALKBH5 targeting group with PM_2.5treatment（P<0.05）.6.The expression of FIP200,ULK1,Atg13 and LC3 in KO-CA group were1.46-fold,1.60-fold,1.17-fold and 1.98-fold higher than that in WT-CA group,relatively（P<0.05）.In comparison with WT-CA group,the levels of NF-κB,NLRP3,Pro-caspase 1,Caspase 1 and IL-1βprotein had a 1.5-fold,1.16-fold,1.30-fold,2.41-fold,and 1.59-fold increase in KO-CA group,relatively（P<0.05）.According to the pathological results of lung tissues,slight pathological changes were found in WT-CA group whereas obvious inflammatory responses were found in KO-CA group.The score of lung abnormalities was relatively increased 1.2-fold in KO-CA group in comparison with WT-CA group（P<0.05）.In comparison with WT-CA group,Masson staining showed a significant increase of fibrotic area in WT-FA group.Compared to KO-FA group,increased collagen deposition was found in KO-CA group.Relatively,Ashcroft score of fibrotic alterations was increased 1.62-fold in KO-CA group in comparison with WT-CA group（P<0.05）.the expression of collagen I andα-SMA in KO-CA group was 1.45-fold,1.34-fold increase when compared to WT-CA group,respectively（P<0.05）.Summary:After PM_2.5 exposure,Atg13 m RNA methylation at site 767 regulated epithelial inflammation-driven pulmonary fibrosis in an autophagy-dependent manner.It provided target intervention strategies towards PM_2.5-induced pulmonary fibrosis.Conclusion:1.mi RNAs in serum played a role in EMT-mediated pulmonary fibrosis induced by PM_2.5.2.PM_2.5 exposure downregulated the expression of mi R-494-3p,targeted the YTHDF2 m RNA,promoted YTHDF2 to recognize Mettl3-mediated m6A-modified CDH1 m RNA,which induced EMT and pulmonary fibrosis.3.Suppression of ALKBH5-mediated Atg13 m RNA methylation at site767 regulated epithelial inflammation-driven pulmonary fibrosis in an autophagy-dependent manner upon PM_2.5 exposure.