NR6A1 Promotes Glycolysis And Proliferation Of Tumor Cells Through MTOR Signaling

The canceration of cells is often accompanied by changes in metabolism.The rapid proliferation of tumor cells requires the cells to continuously absorb nutrients from the environment and generate a large amount of energy.During this process,the glycolysis capacity of the tumor cells is enhanced and plays a role in promoting cancer.However,the reasons for the enhancement of aerobic glycolysis in tumor cells have not yet been fully elaborated.Therefore,it is of great research significance to explore the regulation mechanism of glucose metabolism in tumors.Nuclear receptor subfamily 6 group member 1(NR6A1)is a reproductive nuclear factor whose function involves multiple biological regulatory functions.Most of the current research on NR6A1 is based on its role as a transcriptional repressor,to explore its role and mechanism in embryonic development and cell differentiation.Although some articles report that NR6A1 can promote the proliferation and migration of prostate cancer cells,and it may be a prostate cancer marker and a new member of the tumor-testis antigen family,but so far there has been little research on it in tumors,and the specific molecular mechanism is unknown.In order to explore the function of NR6A1 in glucose metabolism of tumor,the human cervical cancer cells Hela,intrahepatic cholangiocarcinoma cells TFK1 and lung adenocarcinoma cells A549 were used in present study as experimental materials to carry out the following work:1.NR6A1 promotes the proliferation of Hela,TFK1 and A549 cellsSeven tumor cell lines from different organ types were selected for NR6A1 expression analysis,and three cell lines including Hela,TFK1 and A549 with high NR6A1 expression were selected for subsequent research.After RNA interference of NR6A1 in the three cell lines,CCK8,EDU staining and immunoblotting were used to detect cell proliferation activity and PCNA protein expression.The results showed that after interfering with NR6A1,the expression of PCNA protein in three cell lines was significantly down-regulated and the proliferation ability of the cells was obviously inhibited.It suggested that interference with NR6A1 can inhibit the proliferation of tumor cells.2.NR6A1 enhances the glycolysis of Hela,TFK1 and A549 cellsAfter RNA interference of NR6A1 in the three cell lines,the amount of lactate production,glucose uptake,ATP level,and changes in cell mitochondrial membrane potential were detected at the biochemical level.Immunoblotting was used to analyze the protein expression of key glycolytic enzymes,mitochondrial dynamics-related proteins and glycolysis-related signaling pathways(AMPK,mTOR,P38,ERK1/2).The results indicated that after NR6A1 interference,the glucose consumption and lactate production of three cell lines were significantly reduced,and ATP levels and mitochondrial membrane potential were decreased as well.The key enzymes of glycolysis HK1/2 and mitochondrial cleavage protein DRP1 were significantly down-regulated in three cell lines.The phosphorylation levels of p38 and ERK1/2 protein were significantly down-regulated in Hela and A549 cells,but not significantly changed in TFK1 cells.In three cell lines,mTOR was shown significant downregulation at both total protein level and phosphorylation level after NR6A1 interference.It suggested that NR6A1 plays a regulatory role in tumor glycolysis,and the interference of NR6A1 leads to the inhibition of glycolysis and the dysfunction of mitochondria.3.NR6A1 promotes tumor proliferation by enhancing glycolysis through mTORMHY1485,the mTOR phosphorylation activator,was added to the siNR6A1 treated A549 cells to rescue the function of NR6A1.Then the cell proliferation ability,metabolic level and mitochondrial function were detected.It was found that after adding MHY1485,the phosphorylation level of mTOR was significantly increased.At the same time,glucose uptake,lactic acid and ATP production were improved,and the cell proliferation was restored except the cell membrane potential did not change significantly.Further using the cancer genome atlas(TCGA)data to respectively analyze the correlation between NR6A1 and HK1,HK2,DRP1,mTOR expression in lung adenocarcinoma patients,the results showed that the expression of NR6A1 was significantly positively correlated with DRP1 and mTOR.Kaplan-Meier analysis showed that the survival time of patients with high expression of NR6A1 was significantly lower than that of patients with low expression of NR6A1.In summary,NR6A1 can enhance glycolysis through mTOR signaling,thereby promoting the proliferation of tumor cells.In patients with lung adenocarcinoma,high expression of NR6A1 indicates a poor prognosis,suggesting that it may be a potential target for tumor diagnosis and treatment.