Preliminary Study On The Mechanism Of Glutaminase Promoting Proliferation Of Cholangiocarcinoma Through Wnt/β-catenin Signaling Pathway

Background:Currently,most studies have found highly expressed Glutaminase(GLS)in hepatocellular carcinoma,colorectal cancer,breast cancer,glioblastoma,pancreatic cancer,bone cancer and other tumor tissues,and GLS may be involved in the regulation of tumor proliferation and metastasis.However,the high expression of GLS in Cholangiocarcinoma(CCA)and its regulatory mechanism remain unclear.Objective:We used public databases,transcriptomics,metabolomics and immunofluorescence to explore the expression of GLS in CCA and its potential regulatory mechanisms.Methods:Statistically analyzed The differential expression of the transcriptomic data of cholangiocarcinoma tumors and adjacent tissues in The Cancer Genome Atlas(TCGA)database.The differential expression of GLS in CCA tumor tissues and adjacent tissues was obtained,and the correlation between GLS and tumor immune cells,M2 macrophages and their expression genes(CD163,TGFB1,etc.),Wnt/β-catenin signaling pathway related genes(WNT2,WNT3,CTNNB1,etc.),CCND1,MYC was obtained.At the same time,we collected tumor tissue and paracancer tissue samples of CCA that underwent cholangiocarcinoma surgery in The Second Affiliated Hospital of Nanchang University from July 2022 to December 2022.Transcriptomics and gene set enrichment analysis were used to obtain m RNA expression results of GLS,M2 macrophages and their expressed genes(CD163,TGFB1,etc.),Wnt/β-catenin signaling pathway related genes(WNT2,WNT3,CTNNB1,etc.),CCND1,MYC,etc.The metabolomics of Liquid Chromatograph-Mass Spectrometer(LC-MS)detected the differences of metabolites in tumor and adjacent tissues;The correlation between GLS and transcriptomics,metabolomics and macrophages was explored by combined analysis of transcriptomics and metabolomics.Immunofluorescence further verified the correlation between differentially expressed GLS and macrophage subtypes.The clinical data and data of these patients with cholangiocarcinoma were retrospectively analyzed and followed up.Statistical analysis was performed using R language,SPSS 26.0 and Graphpad Prism 8 software.Results:Based on transcriptomic data analysis of TCGA database,GLS are highly expressed in CCA tumor tissues and low expressed in paracancer tissues.GLS gene was positively correlated with mononuclear macrophages,M2 macrophages and their expression genes CCL2,CD163,CHI3L1,TGFB1,TGM2,STAT6,etc.GLS gene was associated with Wnt/β-catenin signaling pathway.For example,WNT2,WNT3,WNT5 A,WNT7B,WNT8 B,WNT9A,and CTNNB1 are positively correlated.GLS gene was also positively correlated with CCA oncogenes CCND1 and MYC.Further analysis showed that WNT1,WNT2,WNT5 A,WNT7A,WNT9 B,WNT10B,CTNNB1 were positively correlated with CD163 gene expression.WNT2,WNT5 A,WNT7A,WNT10 A,WNT10B,CTNNB1 and TGFB1 gene expression were positively correlated;WNT1,WNT4,WNT7 A,WNT11 and CTNNB1 were positively correlated with MRC1 gene.WNT4 and WNT7 B were positively correlated with CCL2 gene.WNT2 and WNT11 were positively correlated with CHI3L1.WNT3 and WNT8 B are positively correlated with STAT6.WNT2,WNT7 B and WNT9 A are positively correlated with TGM2.Transcriptomic analysis showed that the overall gene expression level of tumor tissues in intrahepatic Cholangiocarcinoma(i CCA)was significantly different from that of paracancer tissues(Pearson correlation=0.0894).These differential genes stimulate the body to produce immune response,coagulation response,organ tissue regeneration and other biological processes by regulating macrophage differentiation,phagocytosis,fat and amino acid metabolism,etc.Further analysis showed that GLS gene was highly expressed in CCA tumor tissues,but low expressed in paracancer tissues(P=0.0056).In the i CCA subgroup,GLS gene was highly expressed in i CCA tumor tissue,but low expressed in paracancer tissue(P=0.0164).CCND1 was highly expressed in tumor tissues(P=0.0485),and CD86 was highly expressed in paracancer tissues(P=0.0274).Metabolomics detection showed that there were significant differences in the contents of various metabolic substances between tumor tissues and adjacent tissues in CCA.Although glutamine and alpha-ketoglutaric acid were not statistically different in CCA tumor tissue and paracancer tissue,it was found that the content of glutamine in tumor tissue was higher than that in paracancer tissue.Transcriptomic and metabolomic analysis of 14 CCA surgical specimens showed that GLS gene was positively correlated with WNT2(r=0.44,P=0.0334),WNT3(r=0.59,P=0.00262),and CTNNB1(r =0.83,P=5.61*10-7),was positively correlated with MYC(r=0.69,P=0.000184).CIBERSORT method showed that GLS was positively correlated with M2 macrophages(r=0.44,P=0.03).The results of immunofluorescence showed that the overall expression levels of CD68+,CD86+ and CD163+ in tumor tissues were higher than those in adjacent tissues(P=0.0178).The expression of CD68+ in tumor tissues was higher than that in adjacent tissues(P=0.0495).The expression level of CD163&CD68+ in CCA tumor tissues was higher than that in paracancer tissues(P=0.0437).The ratio of CD86+ to CD68+(i.e.,the ratio of CD86+ to CD68+)was lower in bile duct carcinoma than in paracancer tissue(P=0.0146).The ratio of CD163+ to CD68+(i.e.,the ratio of CD163+ to CD68+)was higher in bile duct carcinoma than in paracancer tissue(P=0.0109).GLS gene expression was positively correlated with the number of CD68+ fluorescent labels(r=0.5799,P=0.0481),and with the number of CD68+&CD163+ fluorescent labels(r=0.7335,P=0.0066).Receiver Operating Characteristic curve(ROCcurve)showed that the Area Under Curve(AUC)was 0.819(95%CI 0.645-0.994).The sensitivity and accuracy of GLS in CCA diagnosis were 91.7% and 66.7% respectively.Subgroup analysis showed that GLS expression level was significantly higher in HBs Ag negative patients than in positive patients(P=0.011),and GLS expression level in CEA < 5ng/m L group was significantly higher than that in CEA≥5ng/m L group(P=0.011).Conclusions:Based on the above results,we speculated that GLS may indirectly promote the polarization of M2 macrophages through metabolic processes,and enable M2 macrophages to secrete WNT molecules to participate in the activation of downstream oncogenes in the Wnt/β-catenin signaling pathway to promote the proliferation of bile duct carcinoma.The difference in GLS expression may provide potential clinical prognostic value for CCA.It may be used as a new molecular target for CCA therapy in the future.