Exploration Of The Omics Mechanism Of TDAG8 Promoting The Survival Of Lung Cancer Cells In Acid Environment

Various types of tumor cell cancer including lung cancer are under a unique pathological microenvironment.This local environment is characterized by inflammation and acidity,and it is conducive to immune escape and drug resistance of the tumor.It is a common focus of biology and medicine to find therapeutic targets based on the characteristics of the tumor microenvironment.Our research group has found that the proton-sensing G protein-coupled receptor TDAG8 could be induced and activated in an acidic environment,resulting in the improved viability of tumor cells.However,the detailed mechanism of this procedure is rarely understood to this date.In this work,non-small cell lung cancer cells were employed as a cell model to explore the histological mechanism of TDAG8 for the lung cancer cell acidic microenvironment survival behavior.In short,TDAG8 stable transfected cells were successfully constructed and then they were stimulated in the acidic medium.After then,these cells were tested by joint metabolomics,transcriptomics analysis and obtained the differential metabolites and genes.After that,the probability of using the TDAG8 and its signaling pathway complex as a target for lung cancer treatment was analyzed.In short,a stable TDAG8 lung cancer cell line was successfully constructed by the lentivirus transfection method.The Real-Time PCR method was used to detect the expression of TDAG8 mRNA,the MTT method was utilized to detect the viability of lung cancer cells and the ELISA kit was employed to detect the lactic acid content in lung cancer cells.The metabolomic analysis was performed by the ultrahigh performance liquid chromatography quadrupole time-of-flight mass spectrometry.and the NovaSeq 6000 sequencing platform is employed for transcriptome sequencing analysis.The GO and KEGG enrichment and other analysis methods are used to analyze the obtained results.The experimental results are list as follows:?1?After the TDAG8 transgenic cells were stimulated in an acidic environment at pH 6.8for 6 h,the expression of TDAG8 in A549-TDAG8 cells was significantly higher than that of A549-vector cells.?2?The survival rates of A549-TDAG8 cells was significantly higher than that of A549-vector cells under the acidic environment of pH6.8.?3?Under the acidic environment of pH6.8,the lactic acid content in A549-TDAG8 cells was significantly lower than that in A549-vector cells.?4?A total of 70 differential metabolites were detected by metabolomics sequencing,including27 up-regulated and 43 down-regulated differential metabolites and they were enriched in 17 metabolic pathways.?5?A total of 357 significant differential genes were detected by transcriptomics sequencing,including 199 up-regulated and 158down-regulated differential genes,and they were enriched in 16 significant metabolic pathways by KEGG.?6?Through GPCR-related analysis of cell survival signaling pathways,the TDAG8 may pass the MAPK signaling pathway,Ras signaling pathway,Ca²⁺signaling pathway,phospholipase D signaling pathway,PI3K-AKT signaling pathway,cAMP signaling pathway,and NF-?B signaling pathway to promote the survival of lung cancer cells.?7?Through the joint analysis of metabolomics and transcriptomics,TDAG8 may pass amino sugar and nucleotide sugar metabolism,glycolysis or gluconeogenesis,lysine biosynthesis,fructose and mannose metabolism,?-linolenic acid metabolism,arginine and proline metabolism,glutathione metabolism,pentose phosphate pathway,niacin ester and nicotinamide metabolism,pyrimidine metabolism and purine metabolism to promote the survival of lung cancer cells.In conclusion,we have demonstrated that the proton-sensing receptor TDAG8 is induced and activated in an acidic environment to promote the survival of lung cancer cells.Based on the omics analysis,MAPK signaling pathway,Ca²⁺signaling pathway,Ras-PI3K-AKT signaling pathway,cAMP signaling pathway and NF-?B and other signaling pathways and amino sugar and nucleotide metabolism,glycolysis,glycogen metabolic pathways,pentose phosphate pathway,nicotinate and niacinamide metabolic pathways may be involved in promoting the survival of lung cancer cells.Most importantly,TDAG8-related signal transduction pathways and metabolic pathways for the survival of lung cancer cells were determined.We target the acidic microenvironment of tumors to provide a new target for lung cancer treatment strategies.