Mechanism Of Orphan Nuclear Receptor NR6A1 Regulating Glucose Metabolism In Ntera-2 Cells

Compared to normal cells,the metabolic pattern of tumor cells has changed dramatically.Normal cells acquire energy primarily through oxidative phosphorylation pathways,whereas tumor cells acquire a large amount of energy mainly through glycolytic pathways.This metabolic pattern of tumor cells is termed the Warburg effect and has become a typical hallmark of tumors.Although Warburg effect is currently considered to be regulated by a variety of genes including transcription factors,such as P53?c-myc and ERR?.However,the transcriptional regulation mechanism of the Warburg effect is still not clear.NR6A1(nuclear receptor subfamily 6 group memb er1,NR6A1)is one of the members of the orphan nuclear receptor family.It is a transcription inhibitor with a specific recognition sequence and can specifically bind any DR0 site.Studies have shown that NR6A1 has the function of promoting the differenti ation of embryonic stem cells and is involved in regulating spermatogenesis and oogenesis.For example,the regulation of NR6A1 on differentiation of embryonic stem cells is mainly achieved through direct inhibition of the pluripotency gene Oct4 at the DR0 site.The experiments in mouse model showed that Nr6a1 participates in the regulation of testicular energy metabolism by inhibiting the expression of mitochondrial glycerol-3-phosphate dehydrogenase,suggesting that it may be related to cellular metabolic behavior.In order to explore the role and mechanism of NR6A1 in tumor cell metabolism,by using human testicular teratoma cell Ntera-2 as the research material,this study carried out the following four aspects:1.Analyzing the effect of NR6A1 on glucose metabolism in Ntera-2 cellsIn Ntera-2 cells,RNA interference the expression of NR6A1 and transfected the NR6A1 plasmid in Ntera-2 cells respectively,and then detected the metabolism-related parameters of cells,such as cellular glucose uptake and lactate production.The results showed that knockdown of NR6A1 promoted glucose uptake and ATP levels in Ntera-2 cells,but lactic acid production was inhibited.And there was no significant effect on ROS content.Overexpression of NR6A1 inhibited glucose uptake and ATP levels in Ntera-2 cells,and promoted lactic acid production,whereas did not change ROS level.2.Analysis of the regulation of metabolism-related genes by NR6A1Bioinformatics software was used to analyze DR0 elements in the promoters of 148 metabolic genes,and there were 12 metabolic genes containing DR0 elements on the promoter were selected.Further,by RNA interference or overexpression of NR6A1 in Ntera-2 cells respectively,the results of qPCR experiments showed that NR6A1 could inhibit the expression of GOT2,HK1,and HK2.Western blot experiments further confirmed that GOT2 and HK1 expression were significantly up-regulated after knockdown of NR6A1.The results showed that NR6A1 exerts an inhibitory effect on GOT2 and HK1.3.Analysis of direct targeting inhibition of GOT2,HK1 and HK2 by NR6A1GOT2,HK1 and HK2 promoter luciferase reporter vectors were constructed,and the effect of NR6A1 on GOT2,HK1 and HK2 promoter activity was analyzed by reporter gene assay.The results showed that compared with the pcDNA3.1 group,the significant decrease of the luciferase activity for GOT2,HK1,and HK2 promoters were investigated in pcDNA3.1-NR6A1 group.Subsequent chromatin immunoprecipitation(ChIP)experiments further confirmed that NR6A1 directly targets GOT2,HK1 and HK2.4.To investigate the role of NR6A1 in the regulation of glucose metabolism by inhibiting the target gene GOT2Both GOT2 and NR6A1 expression plasmids were transfected into Ntera-2 cells respectively,then glucose uptake,lactate production,ATP level,and ROS content were measured.It showed that compared with the pcDNA3.1 control group,glucose uptake and ATP levels were significantly up-regulated in the pcDNA3.1-GOT2 group,and lactic acid production was significantly down-regulated.The results indicated that the effect of GOT2 and NR6A1 on metabolic level in cells is reciprocal.Through functional rescue experiments,the GOT2 plasmid and the NR6A1 plasmid were co-transfected into Ntera-2 cells,and the lactic acid production,glucose uptake,ATP,and ROS levels of Ntera-2 cells were measured.It was found that GOT2 overexpression can abate the inhibition of NR6A1 on cell glycolysis,but can not reduce the inhibition of NR6A1 on ATP levels.The results showed that GOT2 might be a downstream target gene of NR6A1 on inhibiting glycolysis in Ntera-2 cells.In summary,this study preliminarily confirmed that NR6A1 inhibits glucose metabolism in tumor cells by directly targeting GOT2 expression,thereby participating in regulating the occurrence and development of tumor cells.