Multi-omics And Susceptibility Study On Occupational Noise-induced Hearing Loss

Background and objectivesNoise-induced hearing loss(NIHL)is the second most common sensorineural hearing disorder in humans.NIHL has become the second most harmful occupational disease in China,accounting for about one-sixth of the annual increase in occupational diseases,seriously influencing the physical and mental health of workers.NIHL is a complex disease,and its exact pathogenic mechanisms are not fully understood.More and more evidence show that the abnormal expression of proteins and metabolites is closely associated with the occurrence and development of diseases.Proteomics and metabolomics have great potential in describing the overall change characteristics of proteins and endogenous metabolites in the process of diseases occurrence,clarifying the pathogenesis,and searching for biomarkers.There are obvious individual susceptibility differences in the occurrence of NIHL,the contribution rate of genetic factors to the occurrence of NIHL exceeds 50%under the stable noise working environment.Nowadays,although some progress has been made in the research of susceptibility to NIHL,however,most of the single nucleotide polymorphisms(SNPs)studied were not functional,which might not be the genetic variation directly involving in the regulation of the susceptibility to NIHL,therefore,it is difficult to reveal the real cause of NIHL susceptibility and cannot be used as the genetic markers for the diagnosis of NIHL susceptibility.Based on the background mentioned above,this study aims to analyze the present situation and influencing factors of NIHL of workers who exposed to noise in the key occupational disease monitoring enterprises in Jiangsu using cross-sectional survey.Proteomics and metabolomics techniques were used to analyze the differences of expression profiles of protein and endogenous metabolite in the plasma samples of NIHL workers and normal hearing workers,and screen differentially expressed proteins,endogenous metabolites,and abnormal signal pathways related to the occurrence and development of NIHL.Based on the NIHL mouse model,proteomics and metabolomics techniques were used to analyze the differences of protein and metabolite expression profiles in the cochlear tissues of NIHL mice and normal hearing mice,and further explore the key differentially expressed proteins,metabolites,and abnormal signal pathways of NIHL.To screen the same differentially expressed proteins in the plasmas of NIHL workers and the cochlear tissues of NIHL mice and to explore its functional roles and potential molecular regulatory mechanisms in NIHL.Moreover,to screen the functional SNPs of key differentially expressed proteins or key regulatory genes in abnormal signal pathways of NIHL and to explore its relationships with susceptibility to NIHL and potential molecular mechanisms.Methods1.The occupational health examination data of 77990 noise-exposed workers from the key occupational disease monitoring enterprises in Jiangsu during 2015-2020 were collected to analyze the prevalence of NIHL among noise-exposed workers.The gender,age,noise exposure time,noise exposure level,smoking and alcohol consumption were divided into different subgroups and the prevalence of NIHL of noise-exposed workers was analyzed,and multivariate logistic regression analysis was performed to evaluate the strength of the association between each variable and the risk of NIHL.2.TMT-labeled quantitative proteomics was conducted to analyze the differences of protein expression profiles in plasma samples from 3 NIHL workers and 3 normal hearing workers,and non-targeted HPLC/Q-TOF-MS metabolomics was performed to analyze the differences of metabolic expression profiles in plasma samples of 62 NIHL workers and 62 normal hearing workers,and to screen differentially expressed proteins,and endogenous metabolites of NIHL.The candidate differentially expressed proteins POU4F3,ATG5 and COLEC11 were verified by Western blot.RT-q PCR was used to detect the expression levels of PIK3CA,AKT2,Beclin1,ATG5 genes in the peripheral blood of workers,and Elisa was performed to detect the levels of SOD,GSH-Px,IL-6 and TNF-αin the plasma of workers.3.Male C57BL/6 mice aged 6-8 weeks were selected as experimental objects and divided into NIHL group and control group.The mice in NIHL group were exposed to 120 d B SPL broadband white noise for 4 h,while the control mice were not exposed to noise to construct the mouse model of NIHL.Auditory brainstem response(ABR)was carried out in mice,the cochlear tissues of mice were extracted for cochlear basement membrane preparation and immunofluorescence staining analysis.Based on the NIHL mouse model,TMT-labeled quantitative proteomics was used to analyze the differences of protein expression profiles in cochlear tissues of 6 NIHL mice and 6 normal hearing mice,and non-targeted HPLC/Q-TOF-MS metabolomics was performed to analyze the differences of metabolic expression profiles in cochlear tissues of 5 NIHL mice and 5 normal hearing mice,to screen the differentially expressed proteins,metabolites,and signal pathways of NIHL.Western blot was used to verify the candidate differentially expressed proteins ITGA1,KNG1,CFI,FGF1,AKT2,ATG5 and POU4F3.The levels of SOD,MDA,IL-6 and TNF-αin cochlear tissues of mice were measured by Elisa.Western blot,frozen section and immunofluorescence staining were used to analyze the expression and localization of SOD1 and SOD2 proteins in cochlear tissues of mice.4.Mouse cochlear hair cell lines HEI-OC1 were treated using LPS at different concentrations(0、0.25、0.5、1、10μg/m L)at different times(24,48,72 h)to construct the inflammatory model of HEI-OC1 cells.Cell proliferation activity was determined with CCK-8,cell apoptosis and reactive oxygen species(ROS)level were detected by flow cytometry and immunofluorescence staining,and the levels of IL-6 and TNF-αin the supernatant of cell culture were detected by Elisa.Western blot was used to detect the expression change of POU4F3,ATG5,LC3B,p62,Bax,and Bcl-2 proteins in LPS treated cells.Western blot was also choosed to determine the expression levels of LC3B,p62,Bax and Bcl-2 proteins in the cells treated with autophagy activator Rapamycin(RAP)and autophagy inhibitor 3-MA.The HEI-OC1 cell lines with POU4F3 and ATG5 knockdown were constructed,the apoptosis and ROS level,and the expression levels of LC3B,p62,Bax,and Bcl-2 were detected by flow cytometry,immunofluorescence staining and Western blot.5.The occupational health examination data of workers exposed to noise in key occupational disease monitoring enterprises in Jiangsu were collected and divided into NIHL group and normal hearing control group.Based on NCBI db SNP,1000Genomes and Hap Map databases,functional SNPs of autophagy related genes PIK3CA,PIK3R1,PIK3R3,AKT1,ATG4,ATG5 and ATG7 were screened based on the minimum allele frequency(MAF)>0.01 and the linkage disequilibrium r~2>0.8,and the multiplex PCR combined with second-generation sequencing was then performed for genotyping.The bioinformatics database(TFtarget,Func Pred),site mutation,plasmid construction,RNA interference,cell transfection,dual-luciferase reporter assays,RT-q PCR,and Western blot were conducted to explore the potential molecular regulatory mechanism of PIK3R3 SNP rs7536272,ATG5 SNP rs510432,transcription factor SP1 and C/EBPβinvolved in the process NIHL.Results1.Survey on NIHL status of noise-exposed workers and influencing factors analysisThe average noise exposure level of 77990 noise-exposed workers was 87.79±7.24 d B(A),26650 workers were diagnosed with NIHL,the prevalence of NIHL was 34.17%.The prevalence of NIHL was significantly increased in male,aged>35 years,noise exposure time>5 years,noise exposure level>85 d B,smoking and drinking workers(P<0.001).Logistic regression analysis showed that male,age>35 years,noise exposure time>5 years,noise exposure level>85 d B(A),smoking and drinking status were associated with the risk of NIHL and were risk factors affecting NIHL.2.Plasma proteomics and metabolomics of NIHL workers2.1 Plasma proteomics of NIHL workers based on TMT-labeling quantitative techniqueAccording to fold change(FC)>1.2 and P<0.05,a total of 22 differentially expressed proteins were screened between the NIHL group and control group.KEGG pathway enrichment analysis results showed that the differentially expressed proteins were significantly enriched in signaling pathways related to the immune inflammation,autophagy,protein synthesis and processing,and glucose metabolism.Western blot results demonstrated that the expression of ATG5 and POU4F3proteins in the plasma of NIHL workers was significantly decreased in relation to the controls(P<0.05),the expression of COLEC11 increased remarkablely in relation to the control group(P<0.05).2.2 Plasma metabolomic of NIHL workers based on HPLC/Q-TOF-MS techniqueAccording to variable important for the projection(VIP)>1 and P<0.05,a total of 20 different metabolites were identified between the NIHL group and control group.KEGG pathway enrichment analysis demonstrated that differential metabolites were significantly enriched in the metabolic pathways related to phospholipid metabolism,amino acid metabolism,and autophagy.The results of RT-q PCR showed that the m RNA expression of PIK3CA,AKT2,Beclin1 and ATG5 genes in peripheral blood of NIHL workers was obviously decreased compared with the controls(P<0.05).The results of Elisa revealed that the SOD and GSH-Px levels in plasma samples of NIHL workers were significantly lower than those of control group(P<0.01),while IL-6 and TNF-αlevels were obviously higher than that of control group(P<0.001).3.Proteomics and metabolomics of mice cochlea tissues based on NIHL model3.1 Construction of NIHL mouse modelIn relation to the control group,the ABR thresholds of mice in NIHL group were significantly increased at test frequencies of 4,8,12,16,24 and 32 k Hz(P<0.001).The number of outer hair cells(OHCs)in the middle and basal segments of the cochlea in NIHL group mice was significantly lower than that in the control group(P<0.01).3.2 Proteomic study of NIHL mice cochlea tissue based on TMT labeling quantitative techniqueAccording to FC>1.2 and P<0.05,totally,221 differentially expressed proteins were identified between the two groups,with 110 up-regulated proteins and 111 down-regulated proteins.KEGG pathway enrichment analysis revealed that differentially expressed proteins were significantly enriched in signaling pathways related to immune inflammation,protein synthesis and processing,cell proliferation,differentiation and apoptosis,autophagy,and glucose metabolism.Western blot results showed that the protein expression of ITGA1,KNG1 and CFI in the cochlear tissue of NIHL mice was significantly increased compared to the control group(P<0.05),while the protein expression of FGF1,AKT2,ATG5 and POU4F3 was significantly decreased compared to the control group(P<0.05).3.3 Metabolomic study of NIHL mice cochlea based on HPLC/Q-TOF-MS techniqueBased on VIP>1 and P<0.05,a total of 86 differential metabolites were identified between the two groups,of which 48 metabolites were up regulated and 38 metabolites were down regulated.KEGG pathway enrichment analysis results demonstrated that the identified differential metabolites were prominently participanted in the metabolic signaling pathway related to glucose metabolism,amino acid metabolism,phospholipid metabolism,and autophagy.Results of immunofluorescence staining,and Western blot showed that SOD1 and SOD2 were specifically expressed in the cochlear spiral ganglion of mice,and the expression of SOD1 and SOD2 in the cochlea of NIHL mice was significantly decreased than that of control group(P<0.05).Elisa results showed that the SOD activity in the cochlea of NIHL mice was significantly lower than control group(P<0.01),MDA,TNF-αand IL-6 levles were notably higher than ontrol group(P<0.05).4.Study on the molecular mechanism of POU4F3 and ATG5 in the inflammation of HEI-OC1 cells4.1 Construction of LPS-induced inflammatory injury model of HEI-OC1 cellsCCK-8 results demonstrated that in relation to the control group(0μg/m L),the proliferation activity of HEI-OC1 cells was significantly decreased after 48 h treatment with 1μg/m L LPS(P<0.001)and aggravated with the increase of LPS concentration and time.The results of Elisa revealed that the TNF-αand IL-6 levels in the supernatant of cell culture treated with 1μg/m L LPS for 48 h were significantly higher tcompared to the control group(P<0.01).Flow cytometry and ROS detection results showed that the cell apoptosis and ROS levels in the group treated with 1μg/m L LPS for 48 h were significantly higher than those in the control group(P<0.01).4.2 Molecular regulation mechanisms of POU4F3 and ATG5 in the inflammatory injury of HEI-OC1cellWestern blot results show that,POU4F3 protein expression significantly decreased with the increase of LPS concentration(P<0.001).The expression of ATG5 and LC3-II proteins firstly increased and then decreased with the increase of LPS concentration,and the expression level reached the highest under the treatment of 1μg/m L LPS(P<0.001).Compared to the control group,the expression of Bax and Cleaved Caspase-9 proteins was obviously increased after treatment with 1μg/m L and 10μg/m L LPS(P<0.001),Bcl-2 protein expression was significantly reduced(P<0.001).LC3-II and Bax protein expressions in LPS+3-MA group were significantly decreased compared with the LPS group(P<0.001),while p62 and Bcl-2 protein expression were significantly increased compared with the LPS group(P<0.001).Compared to the LPS group,LC3-II and Bax proteins expressions in LPS+RAP group were dramatically increased(P<0.001),the expression of p62 and Bcl-2 proteins was significantly decreased(P<0.001).The expression levels of LC3-II and Bax proteins in the LPS+si-POU4F3 group were dramatically increased compared to the LPS group(P<0.001),the expression of p62 and Bcl-2 was significantly decreased conpared to the LPS group(P<0.01).The results of flow cytometry and ROS revealed that compared to the LPS group,apoptosis and ROS levels were significantly increased in the LPS+si-POU4F3 group(P<0.01).Compared to the LPS group,the expression of LC3-II,ATG5 and Bax proteins in LPS+si-ATG5 group were significantly decreased(P<0.001),the expression of p62 and Bcl-2 was significantly increased(P<0.001).Flow cytometry and ROS results showed that the levels of apoptosis and ROS in the LPS+si-ATG5 group were clearly decreased when compared to the LPS group(P<0.01).5.Study on the relationship between PIK3R3 and ATG5 gene polymorphisms and NIHL susceptibility and its mechanisms5.1 Relationship of PIK3R3 and ATG5 gene polymorphisms and NIHL susceptibilityA total of 688 NIHL workers were recruited as the NIHL group and 667 normal hearing workers were recruited as control group,there were no significant differences in the distribution of age,gender, noise exposure time,noise exposure level,smoking and drinking status between the the two groups(P>0.05).The binaural high frequency threshold on average(BHFTA)in NIHL group was significantly higher than that in control group(P<0.001).PIK3R3 rs7536272 and ATG5 rs510432were significantly associated with NIHL(P<0.05),the rs7536272 G allele significantly increased the risk of NIHL(adjusted OR=1.22,95%CI=1.03-1.45),the rs510432 T allele significantly decreased the risk of NIHL(adjusted OR=0.77,95%CI=0.66-0.90)。5.2 Study on the mechanism of PIK3R3 and ATG5 gene polymorphism affecting NIHL susceptibilityRT-q PCR results showed that the m RNA expression level of PIK3R3 gene in the peripheral blood of individuals with rs7536272 GG genotype was significantly lower than that of individuals carrying AA/AG genotype(P<0.01),the m RNA expression level of ATG5 gene in peripheral blood of individuals with rs510432 CT/TT genotype was significantly higher than that of individuals carrying CC genotype(P<0.01).Dual-luciferase reporter gene results showed that the luciferase activity of HEI-OC1 cells co-transfected rs7536272 wild A allele and SP1 overexpression plasmid was significantly increased compared with the control group and mutant G allele and SP1overexpression plasmid co-transfected group(P<0.001).The luciferase activity of rs510432 mutant T allele transfected with C/EBP-βnegative control plasmid was significantly increased compared to the wild C allele transfected with C/EBP-βnegative control plasmid(P<0.001);the activity of luciferase in mutant T allele and C/EBPβoverexpression plasmid co-transfected group was significantly decreased compared with mutant T allele and C/EBPβnegative control plasmid co-transfected group(P<0.001).Western blot assay demonstrated that compared to the negative control group,the expression of LC3-II,Caspase-3,and Bax protein in the si-SP1 transfection group was significantly increased(P<0.01),while the expression of p-PI3K/PI3K,p-AKT/AKT,and Bcl-2proteins was significantly decreased(P<0.01).The expression of ATG5,LC3-II and Bcl-2 protein in si-C/EBPβtransfection group was significantly increased compared to the negative control group(P<0.05),the expression of Caspase-3 and Bax protein was significantly decreased in relation to negative control group(P<0.05).Conclusion1.The prevalence of NIHL in the noise-exposed workers was higher,male,age,noise exposure time,noise exposure level,smoking and drinking were closely related to the risk of NIHL and were risk factors influencing NIHL.2.The abnormal expression of ATG5 and POU4F3 was involved in the occurrence and development of NIHL and may be potential biomarkers of NIHL.3.Immune inflammation,oxidative stress and autophagy played key roles in NIHL and may be important pathophysiological mechanisms of NIHL.4.Autophagy could promote apoptosis in LPS-induced inflammation of HEI-OC1 cells,and LPS could induce autophagic death of HEI-OC1 cells.5.POU4F3 and ATG5 could influence apoptosis by regulating autophagy activity in the process of LPS-induced HEI-OC1 cell injury,thus affecting the sensitivity of cells to inflammation.6.PIK3R3 rs7536272 and ATG5 rs510432 were associated with susceptibility to NIHL and may be potential susceptible biomarkers of NIHL;Transcription factors SP1 and C/EBPβwere involved in the NIHL by regulating autophagy and apoptosis pathways,which may be potential therapeutic targets for NIHL.