A Preliminary Study On The Mechanism Of Glutaminase Promoting The Proliferation Of Hepatocellular Carcinoma Through Notch Signaling Pathway

Objective:The prognosis of hepatocellular carcinoma(HCC)is poor,and the mechanism of its occurrence and development is extremely complex,which has not been fully elucidated.Previous studies have suggested that glutaminase(GLS)is abnormally highly expressed in hepatocellular carcinoma,which is associated with poor prognosis of patients.Based on transcriptomics and metabolomics,the aim of this study is to explore the possible mechanism of abnormally high expression of GLS in promoting the proliferation of hepatocellular carcinoma,which provide new ideas for the prevention and treatment of hepatocellular carcinoma.Methods:Eighteen cases of liver cancer tumor tissues and adjacent normal tissues of HCC patients who were initially treated and underwent surgical resection in The Second Affiliated Hospital of Nanchang University between July 2022 and December 2022 were collected.The expression of GLS and the concentrations of glutamate,α-ketoglutarate and other metabolites in liver cancer tissues and adjacent normal tissues were analyzed by transcriptomics and metabolomics,and the expression of GLS and M2 macrophages-related specific markers were analyzed.The possible relationship between GLS expression and Notch signaling pathway was found by enrichment analysis,and the correlation between GLS expression and Notch signaling pathway receptors,ligands and downstream target genes was analyzed.Subsequently,the classification and proportion of macrophages in HCC tumor tissues and adjacent normal tissues and their correlation with GLS expression were confirmed by immunofluorescence.Results:Through TCGA transcriptome database analysis and our transcriptome sequencing,it was found that GLS was highly expressed in hepatocellular carcinoma tumor tissues compared with adjacent normal tissues,and the difference was statistically significant(P < 0.05),and the overall survival of patients with high GLS expression was significantly decreased(P=0.052).Untargeted metabolomics found that the expression of glutamic acid and α-ketoglutaric acid related to glutamine metabolism in liver cancer tissues were significantly increased,and the difference was statistically significant(P < 0.05).At the same time,enrichment analysis showed that glutamine metabolism level was significantly increased in hepatocellular carcinoma.Based on TCGA database analysis and transcriptome data,CIBERSORT analysis confirmed that GLS high expression was positively correlated with M2 macrophage infiltration(r=0.394,P < 0.05),and Notch signaling pathway was activated in GLS high expression hepatocellular carcinoma.It was positively correlated with the key molecules and target genes in the Notch signaling pathway(P< 0.05).Immunofluorescence confirmed that macrophages were significantly increased in hepatocellular carcinoma,and mainly M2 macrophages expressed.Pearson correlation analysis showed that GLS expression was strongly correlated with M2 macrophages(r=0.7666,P=0.0036).Conclusion:Based on the combined analysis of transcriptomics and metabolomics,we confirmed that glutaminase was highly expressed in hepatocellular carcinoma,which was negatively correlated with the prognosis of patients.At the same time,abnormally high expression of GLS was positively correlated with the downstream product of glutamine metabolism α-ketoglutaric acid and M2 macrophages in the tumor microenvironment.In addition,the abnormally high expression of GLS was associated with Notch signaling pathway,suggesting that the abnormally high expression of glutaminase in hepatocellular carcinoma promotes significantly increased levels of α-ketoglutaric acid,a downstream metabolite of glutamine metabolism.Alpha-ketoglutaric acid induces M2 macrophages polarization through metabolism and epigenetic reprogramming,and M2 macrophages promotes the proliferation of hepatocellular carcinoma by activating the Notch signaling pathway.This signaling pathway needs to be further confirmed by in vivo and in vitro experiments,and targeting this signaling pathway may provide new ideas for the diagnosis and treatment of hepatocellular carcinoma.