Mass Spectrometry Analysis Of NME2 Related Protein And Signal Network In Liver Cancer Cell HepG2

Background and Objectives:Liver cancer is one of the most common malignant tumors in the world.The continuous increase in morbidity and mortality has caused serious damage to human life and health.The expression of NME2 is closely related to the occurrence of liver cancer.However,its interacting proteins and signal networks in the occurrence and development of liver cancer are not yet clear.Therefore,this study uses trapped ion mobility time-of-flight mass spectrometry to analyze NME2 related proteins and signal networks in liver cancer cells,providing experimental evidence for molecular pathology studies of liver cancer.Methods:(1)Bioinformatics assess the expression level of NME2 in liver cancer and explore the effect of NME2 on the proliferation and colony formation of liver cancer cells: First,assess the expression of NME2 in liver cancer tissues and normal liver tissues through UALCAN software and Oncomine databases.Select Liver cancer cell Hep G2 cells to construct a stable liver cancer cell line that knocks down NME2 in vitro through a lentiviral vector.The experiment is structured in sh-NME2 group and sh-NC group.After the two groups of cells were infected for 72 hours and cultured for 4generations,western blotting was used to detect the expression of NME2 protein in the2 groups of cells;MTT method was used to detect the effect of NME2 on the proliferation of Hep G2 cells;plate and soft agar colony formation experiment was used to detect the effect of NME2 on the colony forming ability of liver cancer cell.(2)Mass spectrometry and bioinformatics analysis of NME2 related proteins and signal networks: In this study,the total proteins of the two groups of Hep G2 were extracted separately.After protein quantification,trypsin digestion,TMT labeling and HPLC classification,the peptides was separated by UPLC and analyzed by Tims-TOF MS.Search related databases based on the obtained difference in protein expression abundance,and the differentially expressed protein molecules are finally obtained,combined with bioinformatics analysis methods,the signal pathways that differentially expressed proteins mainly participate in and the interaction network between the proteins are analyzed.Results:(1)The analysis results of UALCAN software and Oncomine database showed that the expression level of NME2 in liver cancer tissues was significantly higher than that in normal liver tissues,and the difference was statistically significant.After Hep G2 cells stably knocked down NME2,the Western blotting results indicated that the NME2 protein level in the sh-NME2 group was significantly lower than that in the sh-NC group,indicating that the Hep G2 cell line with stable and low expression of NME2 was successfully constructed;MTT results showed that the cell viability in the sh-NME2 group was significantly lower than that in the sh-NC group;The results of plate and soft agar colony formation experiment showed that compared with the sh-NC group,the colony forming ability of the sh-NME2 group were significantly reduced.(2)The results of TMT labeling and mass spectrometry analysis showed that a total of 6656 proteins were identified,of which the difference in the ratio of sh-NME2/sh-NC was 1.3 times as the threshold for differential expression changes,and the t-test P<0.05 was the threshold for significance.Compared with sh-NC,a total of 253 proteins in the sh-NME2 group were up-regulated,and 163 proteins were down-regulated;KEGG enrichment analysis screened out that differential proteins significantly enriched 50 signaling pathways.Up-regulated proteins are mainly involved in NOD-like receptor signaling pathways,steroid hormone biosynthesis,p53 signaling pathways and JAK-STAT signaling pathways,etc,and down-regulated proteins are mainly involved in PPAR signaling pathway,cholesterol metabolism,systemic lupus erythematosus and alcoholism signaling pathways,etc;STRING software analysis results show that NME2 is closely related to 25 proteins in p53,JAK-STAT,PPAR and alcoholism signaling pathways.Conclusion:(1)NME2 protein is highly expressed in liver cancer tissues.After knocking down NME2 in liver cancer cell Hep G2,both the cell proliferation and colony forming ability of liver cancer cells Hep G2 are inhibited.The experimental results are consistent with the previous research results of the research group.NME2 is expected to become a new target for liver cancer treatment research.(2)NME2 may promote the occurrence and development of liver cancer through p53,JAK-STAT,PPAR and alcoholism signaling pathways,and interact with 25 proteins in the four signaling pathways.