| Purposes:Pancreatic cancer is the most malignant tumor in the digestive tract.Most patients with pancreatic cancer are already at an advanced stage when they are diagnosed.It is very important for patients to find new treatment strategies for pancreatic cancer.In recent years,the function of long noncoding RNA(lnc RNA)in tumors has received great attention.The use of next-generation sequencing technology has found that there may be as many as thousands of abnormally expressed lnc RNAs in tumors.Metabolic reprogramming can support tumor cells’ survival and proliferation.The production of α-ketoglutarate(αKG)through the glutamate metabolic pathway is the key to maintaining energy supply and material synthesis.Finding specific lnc RNAs that regulate glutamate metabolism during the tumorigenesis and development of pancreatic cancer,and clarifying their mode of action and mechanism are of great significance to the diagnosis and treatment of pancreatic cancer.Methods:q RT-PCR was used to detect the expression level of lnc RNA XLOC＿006390(named lnc RNA GMA1)in pancreatic cancer cells and pancreatic cancer tissues.The lentivirus-loaded GMA1 overexpression plasmid and sh RNA plasmid were respectively infected with pancreatic cancer CFPAC-1 and Bx PC-3 cells to construct stable transfected cells.Using clony formation assay,Transwell assay,and subcutaneous xenograft models in nude mouse to study the effects of GMA1 expression on the proliferation and migration of pancreatic cancer cells;using targeted metabolomics to detect the changes in key metabolites that are affected by GMA1 expression.The extracellular acidification rate(ECAR)determines whether it affects the glycolysis process;q RT-PCR detects the expression levels of key metabolic genes that GMA1 interferes with,using the luciferase reporter system,plasmid overexpression of the oncogene c Myc,and chromatin immunoprecipitation(Ch IP)assay to clarify whether GMA1 regulates the expression of GDH1 through c Myc transcription;using RNA pull-down assay,constructing GMA1 truncated plasmid,RNA immunoprecipitation(RIP)assay,protein immunoprecipitation(co-IP)assay and ubiquitination detection to determine whether GMA1 functions by directly cross-linking c Myc protein to regulate its ubiquitination level;using immunohistochemistry and bioinformatics analysis to clarify the clinicopathological and prognostic significance of GMA1/c Myc/GDH1 molecular axis in pancreatic cancer.Results:The expression of GMA1 in pancreatic cancer tissues was significantly higher than that in normal tissues.Knockdown of GMA1 inhibits the proliferation and migration of pancreatic cancer cells,while overexpression of GMA1 promotes cell proliferation and migration;overexpression of GMA1 mainly increases the level of αKG in pancreatic cancer cells,while knocking down of GMA1 can reduce the level of intracellular αKG,and GMA1 positively regulates the expression of GDH1 which is the key enzyme for glutamate to synthesize αKG;the oncogene c Myc transcriptionally regulates the expression of GDH1,and GMA1 inhibits its degradation by ubiquitination through targetedly binding with c Myc protein.And GMA1 needs a complete structure for its function;GMA1/c Myc promotes glutamate metabolism and αKG production in pancreatic cancer cells by regulating the expression of GDH1,and promotes cell proliferation and migration;GMA1/c Myc/GDH1 molecular axis is related with the pathological characteristics of pancreatic cancer,and high expression of GDH1 indicates poor prognosis in patients with pancreatic cancer.Conclusion:The GMA1/c Myc/GDH1 axis promotes cell proliferation and migration by regulating the glutamate metabolism and αKG production of pancreatic cancer cells,and the GMA1/c Myc/GDH1 axis is related to the clinicopathological characteristics of pancreatic cancer patients.Therefore,small molecule inhibitors targeting this molecular axis may be a possible way to treat pancreatic cancer from the perspective of interfering with tumor metabolic reprogramming. |
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