The Function And Mechanism Of ADH5 In Non-small Cell Lung Cancer

Background and objective:ADH5(alcohol dehydrogenase 5)is a member of alcohol dehydrogenase family,it’s main function is to metabolize intracellular GSNO(S-nitrosoglutathione)and HMGSH(S-hydroxymethylglutathione).By metabolizing GSNO-a compound formed of active nitric oxide and glutathione,ADH5 indirectly regulates protein S-nitrosylation.ADH5 reduces DNA damage caused by formaldehyde by degrading HMGSH-the spontaneous adduct of formaldehyde and glutathione.ADH5 can promote mitophagy and prevent oxidation.ADH5 knocked out mice are more susceptible to liver cancer induced by carcinogens,indicating the anti-tumor function of ADH5.Yet less is known about ADH5 in lung cancer.Therefore,our study aims to investigate the specific function and possible mechanism of ADH5 in non-small cell lung cancer.Methods:1.By using TCGA database,GEPIA database and kaplan-meier,we analysed the expression of ADH5 in NSCLC and its relation with prognosis.Then we extracted the proteins and RNA of NSCLC cell lines and NSCLC clinical tissues.By performing Western blot assay and qRT-PCR,the expression of ADH5 was detected at two levels.2.The overexpression plasmid and shRNA of ADH5 were applied in NSCLC cell lines H3 58 and PC9 to construct ADH5 stably overexpressed and knocked down cell lines.Then we performed CCK8,clone formation and transwell assay to observe the implication of ADH5 on the proliferation、migration and invasion of NSCLC.Xenograft mouse models were established to confirm the effect of ADH5 on tumor growth in vivo.Immunohistochemistry was applied to detect the expression of Ki67 in xenografts.3.GEPIA database was used to analyze the correlation of ADH5 and SOX2,SOX9.SOX9 protein expressions were analyzed in ADH5 stable cell lines by Western blot assay.TGF-β1 was used to treat ADH5 overexpressed cells to observe the effect of TGF-β1 on cell morphology.Western blot assay was performed to detect the implication of ADH5 on Smad2/3、EMT and AKT pathways.Then we dected LC3 and P62 in stable cell lines after treated with CQ by Western blot assay and immunofluorescence.qRT-PCR was performed to detect the expression of mitochodrial dynamic related proteins.Results:1.Compared with normal lung epithelial cell BEAS-2B,the protein and mRNA expression of ADH5 in H1299 were higher,the mRNA expression of ADH5 in H1975 was higher,the mRNA expression of ADH5 in A549,HCC827,H358,PC9,H460 were lower,the protein expression of ADH5 in A549,HCC827,H358,PC9,H1975,H460 were lower.The downregulation of ADH5 at the protein and mRNA levels in NSCLC clinical tissues were also confirmed.2.Western blot assay and qRT-PCR confirmed the establishment of ADH5 stably overexpressed and knocked down cell lines.CCK8 and clone formation showed that the proliferation of overexpressed cells were inhibited compared with Vector and the proliferation of knocked down cells were enhanced compared with shControl.Transwell assay showed that compared with Vector,the abilities of migration and invasion were enhanced in overexpressed cells,compared with shControl the abilities of migration and invasion were inhibited in knocked down cells.Xenograft mouse models showed that the tumor volumes and weights of overexpression group were smaller than Vector.Immunohistochemistry showed that Ki67 expression of overexpressed group was decreased than Vector.3.ADH5 was found to postively correlated with SOX9 in GEPIA database.SOX9 was upregulated in ADH5 overexpressed cells and downregulated in ADH5 knocked down cells.TGF-β1 was used to treat overexpressed cells for 48h,the morphology of Vector cells significantly changed while overexpressed cells remained unaffected.Western blot showed that ADH5 overexpression promoted Smad2/3 phosphorylation,EMT and AKT phosphorylation were enhanced in overexpression group.Western blot showed that after treated with CQ,LC3 Ⅱ/Ⅰ and P62 in overexpressed cells were higher than Vector,compared with shControl,LC3 Ⅱ/Ⅰ and P62 were lower in knocked down cells.Immunofluorescence assay showed that after treated with CQ,LC3 in overexpressed cells were higher than Vector.qRT-PCR showed that mitochodrial dynamic related protein DRP1 upregulated in overexpressed cells,OPA1 decreased in knocked down cells.Conclusion:1.ADH5 is downregulated in NSCLC at both protein and RNA levels.2.ADH5 promotes migration and invasion but inhibits proliferation of NSCLC,which is associated with tumor cell plasticity.3.ADH5 may regulate NSCLC plasticity through Smad2/3 pathway and autophagy.