The Function And Mechanism Investigation Of GSTM3 In Pancreatic Cancer Malignant Phenotype And Energy Metabolism

BackgroundPancreatic ductal adenocarcinoma(PDAC)is a lethal solid tumor of the digestive system with extremely high malignancy.It is difficult to diagnosis pancreatic cancer early and five-year survival rate is less than 10%.Elucidating the molecular mechanisms of tumor progression is essential to improve patients’outcomes.The glutathione S-transferase(GST)family is a phase II metabolizing enzyme involved in the detoxification of xenobiotic compounds and carcinogens.Among GST family,the distinct roles of glutathione transferase mu-3(GSTM3)in the development of multiple cancers have been elucidated,however,it has not been reported in pancreatic cancer.This study aimed to investigate the relationship between GSTM3 expression levels and the prognosis of pancreatic cancer patients,as well as the regulation of GSTM3 on the malignant phenotype and energy metabolism of pancreatic cancer,to elucidate the potential role of GSTM3 in pancreatic cancer and its downstream molecular mechanisms.MethodsHigh-throughput protein chip Huprot was previously used to screen the disease-related markers of pancreatic cancer and select the target molecule for this study.The tissue microarray of pancreatic cancer was used for immunohistochemical staining to analyze the relationship between the GSTM3 expression level and clinicopathological parameters and prognosis.Stable transfection pancreatic cancer cell lines with GSTM3 overexpression or knockdown were established.Cell counting kit-8 assay,colony formation assay,Transwell and flow cytometry were used to detect the effect of GSTM3 on proliferation,invasion,migration,cell cycle and apoptosis.Next,we constructed the subcutaneous xenograft tumor model in nude mice to assess the malignant biological behavior of pancreatic cancer in vivo.ROS was observed by microscope.Moreover,Seahorse metabolic detection platform was used to explore the regulation of GSTM3 on the energy metabolism of pancreatic cancer cells,including glycolytic capacity and mitochondrial respiration.Then LC-MS/MS was applied to detect targeted energetic metabolites.Finally,the next-generation sequencing was performed to reveal the molecular mechanism of GSTM3 in regulation of malignant phenotype and energy metabolism at the transcriptome level,and to search for differential genes and downstream targets.ResultsGSTM3 was selected by Huprot as significantly less positive in pancreatic cancer than in controls.First,immunohistochemical staining of GSTM3 in 97 human pancreatic cancer tissues showed that the expression level of GSTM3 in tumor tissue was lower than adjacent tissue.Kaplan Meier survival analysis showed that the prognosis of patients with high GSTM3 expression level was significantly better than that of patients with low GSTM3 level.Multivariate regression analysis proved that GSTM3 was an independent risk factor for the prognosis of PDAC patients.Next,four stable transfection pancreatic cancer cell lines(CFPAC-1,BXPC-3,MIA PACA-2,PANC-1)were constructed to biaxially regulate the GSTM3 level,and the transfection efficiency was verified by Western blot and PCR.Functional experiments showed that overexpression of GSTM3 could inhibit the proliferation of pancreatic cancer cells,and knockdown of GSTM3 could promote the cell proliferation.Furthermore,cell cycle detection showed that GSTM3 significantly affected G1/S transition.The variation trend of cell cycle-related proteins detected by WB was consistent with the above results.Based on the result of cell proliferation,the subcutaneous xenograft tumor model in nude mice was established to further verify the previous conclusion in vivo.Except for CFPAC-1 cell line,the results of the other three cell lines were consistent with the in vitro experiments.Using confocal laser microscopy to directly observe the intracellular ROS level labeled with DCFH-DA probe,the results confirmed that GSTM3 could significantly affect the level of ROS,and the ROS level increased significantly by knockdown of GSTM3.Moreover,GSTM3 inhibited glycolysis(rate and capacity)in pancreatic cancer cells by Seahorse energy metabolism detection.In terms of mitochondrial function,PDAC cells with GSTM3 knock-down had higher levels of respiratory capacity and vice versa.Furthermore,LC-MS/MS was used for qualitative and quantitative detection of energy metabolites.Through metabolite identification and quality control,some metabolites with differences,including those related to carbohydrates and amino acids,were obtained.Glucose,L-citrulline,and L-ornithine were significantly elevated in the GSTM3 knockdown PANC-1 cell line compared with controls,and the differences were statistically significant.Finally,using transcriptome sequencing to reveal the molecular mechanisms by which GSTM3 regulates the malignant biological behavior of pancreatic cancer,differential genes were sought and subjected to enrichment analysis,which could be enriched for numerous metabolism related pathways,such as central carbon metabolism,glycolysis/gluconeogenesis,and glutathione metabolism.ConclusionsThe expression level of GSTM3 in pancreatic cancer tissues was lower than that in adjacent cancerous tissues and was an independent indicator for prognosis.GSTM3 inhibited the proliferation of pancreatic cancer cells in vivo and in vitro,which in turn affected tumor progression.GSTM3 can regulate the energy metabolism of pancreatic cancer cells,including the glycolytic capacity and the mitochondrial respiration level,to meet the demand of malignant cells proliferation.Targeting numerous metabolic pathways downstream of GSTM3 may provide a new direction for the treatment of pancreatic cancer.