Knockout Of ENO1 Inhibits The Glycolysis And Tumorigenicity Of Pancreatic Cancer Cells

Objective: Cancer cells produce energy by aerobic glycolysis rather than mitochondrial oxidative phosphorylation even in the aerobic circumstances.Glycolysis provides a large number of substrates to support the vigorous growth of cancer cells,and extracellular acidification induced by glycolysis leads to the immune escape.As one of the key enzymes in glycolysis pathway,ENO1 catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate.It has been widely reported that ENO1 highly expresses in a variety of human cancers,which helps to promote the glycolysis and tumor growth,and is considered to be a potential cancer biomarker and therapeutic target.Nevertheless,the role of ENO1 in abnormal glucose metabolism of pancreatic cancer has not been well studied.The aim of this research was to verify the effects of ENO1 knockout on the biological functions of pancreatic cancer cells,and to further explore the molecular mechanisms and regulatory pathways of ENO1 in abnormal glucose metabolism,which will help to study the pathogenesis of pancreatic cancer and find more effective regulatory targets.Methods: We designed ENO1 knockout sg RNA sequences and constructed the ENO1 knockout expression vectors based on CRISPR-Cas9 technology.PANC-1 and MIA-Pa Ca-2ENO1 knockout stable cell lines were established by lentiviral transduction and the expression of ENO1 was detected by Western Blot.Then we used Cell Counting Kit-8 to detect the change of cell proliferation after the knockout of ENO1 in pancreatic cancer cells.And the effect of ENO1 knockout on the migration and invasion abilities of pancreatic cancer cells was examined by Transwell assay.Colony formation assays in 6-wells plates were used to determine the colony formation rates.We then evaluated the tumorigenicity of pancreatic cancer cells by soft agar assay in vitro and subcutaneous xenograft model in NOG mice.In order to evaluate the change of glycolysis after the knockout of ENO1 in pancreatic cancer cells,cell culture medium of the control groups and ENO1 knockout groups was collected to measure the glucose consumption and lactate production.After the above assays were completed,RNA-seq was conducted to identify the differentially expressed genes in pancreatic cancer cells after the knockout of ENO1,and several differentially expressed genes were randomly selected and the changes of their m RNA expression levels were tested by q RT-PCR to verify the reliability of the sequencing results.GO enrichment analysis was conducted to study the major functions of these genes and the biological processes they involved in,the pathways and differentially expressed genes related to cell metabolism were identified by KEGG pathway analysis.KEGG pathway analysis was conducted to find out the pathways related to cell metabolism and the genes involved in these pathways,so as to further explore the signaling pathways in which ENO1 regulates the abnormal metabolism of pancreatic cancer cells.Results: We established ENO1 knockout PANC-1 and MIA Pa Ca-2 stable cell lines which were verified by Western Blot.In the Transwell assays,we found that the number of pancreatic cancer cells migrated and invaded through the chamber membrane with ENO1 knockout was significantly less than the control groups.The growth curves in the CCK-8 assays showed that the proliferation was significantly inhibited in the ENO1 knockout pancreatic cancer cell,and the colony formation rates of pancreatic cancer cells dramatically decreased after the knockout of ENO1.Tumorigenicity of pancreatic cancer cells evaluated by soft agar assays in vitro was significantly reduced in the ENO1 knockout groups,this was confirmed by the subcutaneous xenograft model in NOG mice,the tumor sizes of the ENO1 knockout pancreatic cancer cells were significantly smaller than the control groups,and the tumor growth curves indicated that the knockout of ENO1 in pancreatic cancer cells inhibits the tumorigenicity.The glucose consumption and lactate excretion in the ENO1 knockout pancreatic cancer cells reduced sharply,suggesting a decreased level of glycolysis.We identified 727 differentially expressed genes between the ENO1 knockout groups and the control groups of pancreatic cancer cells by RNA-seq,which were mainly associated with cellular components such as the extracellular matrix and the endoplasmic reticulum lumen.The differentially expressed genes were functionally related to receptor ligand activity and receptor regulator activity,and involved in biological processes such as heart development and regulation of signaling receptor activity.KEGG pathway analysis found that ENO1 may play an important role in pentose phosphate pathway,fructose and mannose metabolism,amino sugar and nucleotide sugar metabolism,and may affect the expression of G6 PD,ALDOC,UAP1 and other genes in these pathways,it provides a potential way to further study the mechanism of ENO1 knockout in inhibiting pancreatic cancer.Conclusion: The knockout of ENO1 in pancreatic cancer cells inhibits cell proliferation,migration and invasion,it also significantly reduced the level of glycolysis.The knockout of ENO1 may inhibit the tumorigenicity by affecting the expression of G6 PD,ALDOC,UAP1 in the related metabolic pathway.