The Mechanism Of ACLY In Epithelial-mesenchymal Transition Of Lung Airway Epithelial Cells Induced By PM_2.5 Exposure

Recently,epidemiological studies showed the dramatic evidents which comented,fine particulate matter(PM_2.5)with lung tumorigenesis.Epithelial-mesenchymal transition?EMT?has been shown to be involved in the initial process of malignant transformation and tumorigenesis.In this research,we focused on the ATP citrate lyase?ACLY?-mediated energy metabolism in lung EMT induced by PM_2.5 exposure at in vivo and in vitro.It was found that PM_2.5 promoted the EMT process through activating ACLY.On the other hand,inhibiting the expression of ACLY could block the PM_2.5-mediated EMT process in lung.The data showd that inhibition of ACLY protectd airway epithelial cells from EMT induced by long-term PM_2.5exposure,which providing novel clues to figure out the molecular mechanism on PM_2.5-associated lung tumorigenesis.PM2.5 exposure induces epithelial-mesenchymal transition?EMT?in pulmonary cell1.In order to investigate the effects of long-term exposure of PM_2.5 on HBE cells,we prepared the solution of PM_2.5 at a concentration of 0,100,and 500?g/ml.CCK-8 activity assays were used to verify the proliferation of HBE cells on PM_2.5 exposure at for 8,17,26,and35 passages,respectively.The proliferative capacity of HBE cells was increased in a time-dependent manner following PM_2.5 exposure.It is noted that HBE?P35?showed the highest capacity of proliferation?P<0.001?.2.EdU assay showed that the cell viability of the PM_2.5-treated group?P35??100 and 500?g/ml?was significantly higher than that of the control group?0?g/ml??P<0.05?.3.Further,we detected the migration and invasion ability of cells by Transwell assay.The results showed that HBE cells exposed to PM_2.5?P35?showed higher migration and invasion abilities than control?P<0.01?.4.Long-term induction of PM_2.5?P35?showed a significant increase in colony counts in the treatment group compared to the control group in HBE cells?P<0.01?.5.To study PM_2.5-induced tumorigenesis and metastasis in HBE cells,we conduct subcutaneous tumor implantation and tail vein injection in nude mice respectively,and then to investigate whether the malignant tumors of HBE cells?P35?have been malignant after long-term PM_2.5 exposure.The results showed that after implantation of the cells,there was a significant difference in the rate of tumor formation and volume in the ventral region between the treated group and the control group?P<0.01?.After injection into the tail vein,HBE cells were targeted to colonize the nude mice's liver,but no tumor formation were observed in the lungs.6.On both the mRNA and protein levels,qRT-PCR and Western blot were performed to detect pathway factors in the process of EMT.The mRNA expression levels of N-cadherin,Vimentin and Snail were detected after exposure to PM_2.5?P35?in HBE cells and all of them were significantly enhanced.While mRNA expression levels of epithelial markers E-cadherin and ZO-1 were significantly down-regulated.The results of changes in protein expression levels were consistent with those on the RNA levels.7.To further validate PM_2.5-induced EMT,we used dynamic inhalation exposure to detect biochemical changes after PM_2.5 exposure at the animal level?6 male C57BL/6 mice per group,8 hours/day exposure?.After microCT scan,it was found that after 21 days of exposure,alveolitis,emphysema,inflammatory cells infiltration around the bronchus,and fibrosis have been obersed in the murine lung tissues.8.The qRT-PCR method was used to detect the RNA expression levels on the factors in the EMT process in lung homogenates of mice.N-cadherin and Vimentin were significantly up-regulated?P<0.05?,while E-cadherin and ZO-1 were both down-regulated.9.E-cadherin and N-cadherin were selected for immunohistochemical staining?IHC?to observe changes in EMT protein levels in tissues exposed to PM_2.5.The IHC score showed that the expression of E-cadherin in the lungs of exposed mice was significantly down-regulated?P<0.05?,while the expression of N-cadherin was significantly up-regulated?P<0.01?.Metabolomics analysis of HBE cells following long-term PM2.5 exposureIn the above studies,we verified the EMT of airway epithelial cells induced by long-term PM_2.5 exposure and induced malignant transformation of HBE cells.In order to further study the key regulators of PM_2.5-induced EMT,we used metabonomics to screen EMT-related metabolites in cells,and followed with bioinformatics analysis to determine the signaling pathway of the metabolites.1.Using the GC/MS technology for metabolomics analysis,based on the EIC data of more than 200 small molecule compounds established in the database,with FC>1.5 and P<0.05 as the cut-off,18 metabolites were identified?10 up-regulated and 8 down-regulated?.Subsequently,via the on-line tool MetaboAnalyst 3.0,the pathway enrichment of significantly regulated metabolites was analyzed.The results showed that citrate participated in the first two enrichment pathways.2.In order to verify the above deduction of ACLY activation,expression of ACLY were assayed both in vitro and in vivo after exposure to PM_2.5.The results showed that expression of ACLY was up-regulated in both mRNA and protein levels in HBE cells induced with PM_2.5.Consistent with in vivo studies,the expression of ACLY mRNA in lungs was also up-regulated after dynamic inhalation exposure to PM_2.5 in mice.3.As a catalytic product of ACLY,the expression level of acetyl-CoA in cells was determined by ELISA.It was found that after exposure to PM_2.5,its expression was significantly increased in a dose-dependent manner?P<0.01?.ACLY regulates PM2.5-induced lung cell EMTIn order to investigate the role of ACLY in PM_2.5-induced EMT,we constructed a multiple shRNA-interfering ACLY knockdown cell line?ACLY KD?.The with wild-type HBE cell line?HBE WT?and ACLY KD HBE cells were treated by PM_2.5?0 and 100?g/ml?to verify the biological function of ACLY in the progress of EMT.1.The expression of ACLY in the ACLY KD cell lines was verified by qRT-PCR and Western blot.The results showed that the mRNA and protein expression of ACLY KD cells were significantly decreased after shRNA knockdown.2.Detect the changes of EMT related factors in long-term exposure of PM_2.5 HBE WT and ACLY KD cells were detected to verify the effect of inhibition of ACLY expression on the EMT process.The results of qRT-PCR and Western blot showed that the transfection of ACLY shRNA lentivirus inhibited the changes of N-cadherin,Vimentin,Snail,E-cadherin and ZO-1protein levels on the long-term PM_2.5 exposure.3.It was found that after inhibition of ACLY expression,cell activity decreased,which can effectively curb the increase in activity caused by long-term exposure to PM_2.5 with CCK-8 and EdU assays.4.Date from the ell migration and invasion experiments showed that inhibiting the expression of ACLY significantly reduced the migration and invasion capacities of PM_2.5 after long-term exposure?P35??P<0.01?.5.The clony formation assay showed that inhibition of the expression of ACLY reduced the number of colonies induced by PM_2.5 exposure.6.To investigate the effect of ACLY expression inhibition on PM_2.5-induced malignant transformation,HBE cells?P35?were injected subcutaneously and via tail vein in nude mice.The WT and ACLY KD types were implanted into nude mice,and the tumorigenic and metastatic levels of the cells were compared.The results showed that there was a significant difference in the tumor formation rates and tumor volumes between the exposed group and the control group?P<0.01?.Meanwhile,WT type cells showed metastasis in the livers.The volume of tumor formed by ACLY KD cells is significantly smaller than that of WT cells?P<0.01?.We found that inhibiting the expression of ACLY can effectively blunt the metastasis in vivo.In summary,we found that long-term exposure to PM_2.5 induced EMT in HBE cells,as well as changes in cell phenotype and function during the EMT process,which have been verified at the cellular and animal levels.It shows that long-term PM_2.5 exposure can induce EMT in bronchial epithelial cells and promote its malignant transformation.Combined with metabonomics and bioinformatics analysis,we believe that ACLY is a key regulator of PM_2.5-induced energy metabolism in EMT processes.Therefore,by inhibiting the expression of ACLY,we further explore the role of ACLY in the regulation of EMT.It was found that by inhibiting the expression of ACLY,the progression of EMT induced by PM_2.5 can be blocked in HBE cells,and the degree of malignant transformation can be effectively reduced.Therefore,this study suggested that ACLY can be used as a target for the prevention and treatment of PM_2.5 exposure induced pulmonary distorders.