Effect And Mechanism Of Tumor-like Transformation Of Human Bronchial Epithelial Cells Induced By Coal Dust Exposure

Background and purposeCoal is still one of the important energy sources that cannot be replaced.Epidemiological studies have shown that the incidence of lung cancer among coal mine workers is higher than that of the general population,indicating that environmental pollutants in coal mining areas may increase the risk of lung cancer.This study uses human bronchial epithelial cells as a model to study the carcinogenic potential and potential toxicological mechanisms of coal dust(CD)exposure;and provides experimental evidence for the risk assessment and exposure control of coal dust.Methods:(1)Review and summarize the epidemiological evidence for the association between coal dust exposure and lung cancer risk to assess whether coal dust exposure is associated with increased lung cancer risk.(2)Human Bronchial epithelial cells(BEAS-2B)were exposed to coal dust and subcultured.At the cellular level,morphological changes,proliferation activity,migration ability,DNA repair ability and genomic stability of BEAS-2BCD cells after coal dust exposure were detected by hematoxylin-eosin staining(HE),methylthiazolyldiphenyl-tetrazolium bromide(MTT),clone formation assay,scratch-healing assay,indirect immunofluorescence,comet assay,protein immunoblotting and high-throughput sequencing(NGS).Xenotransplantation of BEAS-2BCD cells into BALB/c nude mice was performed to observe the tumorigenic potential of BEAS-2BCD cells in vivo.The tumor-like transformation of BEAS-2BCD cells after coal dust exposure was evaluated comprehensively.(3)BEAS-2B cells were exposed to coal dust for a short time.Stress injury mechanism was explored by HE staining,mitochondrial membrane potential fluorescence probe,reactive oxygen species(ROS)fluorescence probe,indirect immunofluorescence and protein immunoblotting.On the basis of this damage mechanism,protein immunoblotting,cellular immunochemical staining,reverse transcription-polymerase chain reaction(RT-PCR),annexin V-FITC/PI and flow cytometry were used to investigate the possible mechanism of tumor-like transformation of BEAS-2BCD cells induced by coal dust chronic exposure.Results(1)Meta-analysis showed that the Relative Risk(RR)of coal dust exposure and lung cancer was 1.46(95%CI 1.29-1.66),indicating that the lung cancer risk of exposed people was 1.46 times higher than that of the non-exposed people,which suggests that there was a positive correlation between coal dust exposure and lung cancer,and coal dust exposure is a risk factor for lung cancer.(2)Biological characterization of tumor-like transformation of human bronchial epithelial cells(BEAS-2BCD)cultured for 30 generations under coal dust exposure:1)Morphology:BEAS-2BCD cells showed increased volume,enlarged nucleus,abnormal ratio of nucleus to cytoplasm,hyperchromatic nuclei and increased nucleoli,which was significant dissimilation compared with normal BEAS-2B cells.2)Functional characterization:the average viability of BEAS-2BCD cells cultured for 24h,48h,72h,and 96h were 0.64,0.75,0.78,0.85,respectively,which was significantly higher than that of BEAS-2B cells normally cultured for the same generations(0.46,0.65,0.67,0.73)(p<0.05).At the same time,the average clone formation ratio of 31.10%of BEAS-2BCD cells was three times higher than that of BEAS-2B cells(10.13%)(p<0.01).Moreover,the 24h migration rate of BEAS-2BCD cells(50.83%)was significantly higher than that of BEAS-2B cells(17.71%)(p<0.01).These demonstrated that the proliferation activity and migration ability of BEAS-2BCD are significantly enhanced.In addition,the average number of y-H2AX foci(18/cell)of DNA breakage in BEAS-2BCD cells treated with cisplatin for 24h was significantly less than that of the BEAS-2B cells(26/cell)(p<0.05);after removing cisplatin and continuing to culture for 12h and 24h,the average number of y-H2AX foci of BEAS-2BCD was 9/cell and 4/cell,respectively,which were significantly less than that of BEAS-2B cells(22/cell and 15/cell)(p<0.05).Comet experiment showed that the DNA damage rate of BEAS-2BCD cells(15%,8%,3%)was also significantly lower than that of BEAS-2B cells(19%,14%,10%)when cisplatin was treated for 24h and removed for 12h and 24h(p<0.05).Western blotting showed that the phosphorylation levels of DNA repair enzyme ATM/ATR and DNA-PKcs in BEAS-2BCD cells were higher than those of BEAS-2B cells(p<0.05).The above results confirmed that the DNA repair ability of BEAS-2BCD cells was significantly enhanced,suggesting that its adaptability to the environment and anti-apoptosis ability were significantly improved.3)Transcriptional characterization of genome:NGS testing showed that the genome of BEAS-2BCD cells was unstable.The expression of promoting proliferation and migration,and anti-apoptosis related genes(CXCR4,CASC9,CNPY2,CRNDE,FAM83A,EGFR,BCAT1,HMGA1,NSUN2,ANKHD1,ZNF326,etc.)was increased significantly;the expression of proto-oncogenes(MET,MYC,FYN,ABL2,ATF1,SET,ETS2,etc.)was significantly increased,while the expression of tumor suppressor genes(ADARB1,GLIPR1,CDO1,CTDSPL,DLC1,TOM1L2,etc.)was significantly reduced;pathways of cell transmembrane signal transduction(Integrin,EGFR,VEGF,PDGF,etc.)and intracellular signal transduction(PI3K/AKT,MAPK,mTOR,etc.)were significantly up-regulated.The results indicate that BEAS-2BCD cells exposed to coal dust have tumor-like transformation-related biological activities of significantly enhanced metabolic activity,self-renewal,proliferation,migration,angiogenesis,injury repair and anti-apoptosis compared with BEAS-2B cells.4)Characterization of tumorigenesis in vivo:The results of animal experiments displayed that the neoplasms formation rate of BEAS-2BCD cells was 20%and the terminal volume was 726.0mm3.The neoplasms formation rate of the positive control group of lung adenocarcinoma cell line H358 was 60%,and the average volume was 2088.2mm3.HE staining of neoplasms tissues showed that the cells in the BEAS-2BCD group grew mixed,with more tissue cells reactions,and the nuclear heteromorphism was obvious.The positive cells of Ki-67 were high in BEAS-2BCD group and close to H358 positive control group,which indicated that BEAS-2BCD cells had strong proliferative ability.The establishment of BEAS-2BCD cell tumor-bearing model showed that the cells had the potential of tumorigenesis in vivo.(3)The results of mechanism research showed that short-term exposure to coal dust caused mitochondrial membrane depolarization(0h:3.79%,6h:6.45%,12h:17.34%,24h:25.03%).ROS production was increased significantly(0h:4.08;6h:21.13;12h:75.46;24h:30.75);mitochondrial apoptotic pathway protein Caspase-9,Caspase-3 and DFF45 in BEAS-2B cells were activated.Both of them induced DNA breakage effect.The phosphorylation levels of DNA repair enzymes ATM/ATR and DNA-PKcs,as well as their related pathway proteins of AKT,mTOR,p70S6K,4E-BP1,EGFR and ERK in BEAS-2B cells cultured for 10th,20th and 30th generations under coal dust exposed were significantly increased compared with normal BEAS-2B cells,which suggests that DNA break damage activates the two DNA repair pathways of HR and NHEJ and their associated signal pathways AKT/mTOR and EGFR/ERK in coal dust exposed cells.The application of AKT,EGFR,ERK inhibitors(MK-2206 2HCl,Gefitinib,LY3214996)could significantly reduce the proliferation activity,migration ability,inhibit cell cycle progression and promote apoptosis of BEAS-2BCD cells.In addition,ERK inhibitor(LY3214996)significantly decreased the phosphorylation level of the downstream proto-oncogene transcription factors MYC,ATF1 and ETS2.These results suggest that abnormal activation of AKT/mTOR and EGFR/ERK signaling pathway may play an important role in tumor-like transformation of BEAS-2BCD cells.Conclusion and significance(1)Coal dust exposure can increase the risk of lung cancer,is one of the risk factors of lung cancer.(2)Coal dust exposure can induce tumor-like transformation of human bronchial epithelial cells with excessive proliferation,migration,anti-apoptosis,genomic instability and tumorigenic potential in vivo.(3)Coal dust exposure can drive DNA damage through mitochondrial membrane depolarization,ROS generation and activation of mitochondrial apoptotic pathway,activate DNA repair pathway and related signal pathways AKT/mTOR,EGFR/ERK,etc.,which may be one of the important toxicological mechanisms to induce tumor-like transformation of human bronchial epithelial cells.The results of this study provide new experimental evidence for the potential role of coal dust exposure in lung cancer,and provide research ideas for further elucidating the pathogenesis of related lung cancer,which has a certain value of prevention and control of occupational hazards.Figure 26 table 13 reference 214...