The Role And Mechanism Of TMEM100 In Non-Small Cell Lung Cancer

ObjectiveLung cancer is one of the most malignant tumors with the highest morbidity and mortality in China and the world.About 1.6 million people die of lung cancer every year,of which non-small cell lung cancer?NSCLC?accounts for more than 80%.With the increase of smoking and environmental pollution,the incidence of lung cancer is also increasing year by year.NSCLC is a complex heterogeneous disease,and some patients present with molecular changes such as KRAS?TP53?LKB1?EGFR mutation or ALK rearrangment.Although molecular targeted therapies have been used in clinical treatment and have made some achievements,the 5-year survival rate of patients with lung cancer is still lower than 15%due to the late diagnosis,early metastasis,the non-broad-spectrum of molecular targeted drugs and extensive individual differences.Therefore,in-depth study of the molecular mechanisms during NSCLC development and searching for new molecular targets are particularly important for the early diagnosis and treatment of NSCLC.Methods1.NSCLC expression profiles were downloaded from GEO database and differentially expressed genes?DEGs?between NSCLC tissues and control tissues were screened by GEO2R.GO?KEGG and PPI network analysis were used to characterize the DEGs and identify the candidate gene.The expression status of candidate gene was further confirmed in TCGA and CCLE database.Meanwhile,Kaplan-Meier Plotter was applied to analyze the effect of candidate gene on the survival of patients with NSCLC.2.The expression of candidate gene was further verified in NSCLC tissues and cell lines by immunohistochemistry and Q-PCR respectively.After overexpressing and interfering with candidate gene in NSCLC cells,CCK8,colony formation assay and cell cycle analysis were used to explore the effects of candidate gene on the proliferation and cell cycle of NSCLC cells.Flow cytomertry and Western blot were conducted to study the role of candidate gene in cell apoptosis.Wound healing and Transwell experiments were applied to determine the effect of candidate gene on cell migration.3.After overexpression and interference of candidate gene in NSCLC cells,their effects on cell autophagy were detected and the relationship between autophagy and apoptosis was explored.4.Whole transcriptome sequencing was performed on candidate gene overexpressed A549 cells and control cells to provide a guidance for the follow-up mechanism research.5.NSCLC cells which stably express target gene were employed to animal experiments.Results1.23 co-up-regulated genes and 121 co-down-regulated genes were screened from four human NSCLC-related GEO data sets?GSE19804,GSE27262,GSE43458 and GSE18842?.TMEM100 is one of the most significantly down-regulated genes.The absolute values of log₂FC of TMEM100 in the four datasets were greater than 3.GO analysis showed that TMEM100 mainly involved in endothelial cell differentiation and development,blood vessel formation,protein kinase B?PKB?signaling pathway,mesenchymal cell differentiation,epithelial mesenchymal transition?EMT?,BMP signaling pathway.And these biological processes are closely related with the occurrence of tumor development.Meanwhile,the low expression of TMEM100 in NSCLC was also verified in TCGA and CCLE database.Furthermore,the expression of TMEM100 was negatively correlated with the stage of patients and positively correlated with survival rate,indicating that TMEM100 may play a role in NSCLC as a potential tumor suppressor gene.2.Low expression of TMEM100 was also confirmed in human NSCLC tissue samples and cell lines.Overexpression of TMEM100significantly inhibited the proliferation and colony formation of A549 cells.In addition,TMEM100 caused cell cycle arrest and promoted apoptosis of A549 cells.Opposite results were obtained after interfering with TMEM100 in H460 cells.However,overexpression of TMEM100 did not affect the migration of A549 cells compared to the control group.A549cells which stably express TMEM100 and control cells were used to conduct tumor formation experiments in nude mice,and it was found that TMEM100 could inhibit the growth of subcutaneous tumor.3.The ratio of LC3?/LC3?was increased after overexpression of TMEM100 in A549 cells,and p62 was down-regulated by TMEM100.Moreover,TMEM100 significantly inhibited the phosphorylation of PI3K,AKT.Interference of TMEM100 in H460 cells got the opposite results.Furthermore,inhibition of autophagy by Baf-A1 promoted the apoptosis of A549 cells induced by TMEM100.4.The transcriptome sequencing results showed that there were 1767genes up-regulated and 1092 genes down-regulated after TMEM100overexpression?P<0.05,|log₂FC|?1 as cutoff criteria?.GO analysis found that the biological processes enriched by DEGs mainly involved in endoplasmic reticulum stress?ERS?and unfolded protein response?UPR?.KEGG pathway analysis also showed the most significantly enriched pathway was protein processing in endoplasmic reticulum.ERS associated genes such as CHOP,GADD34,PERK were significantly decreased after TMEM100 overexpression.TMEM100 was mainly located in the endoplasmic reticulum and plasma membrane,and overexpression of TMEM100 can significantly inhibit the expression of endoplasmic reticulum stress related proteins,such as CHOP and XBP1 in A549 cells.Interfering TMEM100 in H460 cells obtained the opposite results.These results suggest that TMEM100 may play a role in inhibiting ERS,and the mechanism remains to be further studied.ConclusionIn this study,TMEM100 was selected as a potential tumor suppressor and it was found to inhibit the process of NSCLC by activating autophagy and inhibiting endoplasmic reticulum stress response.