Long Non-coding RNA PVT1 Promotes Warburg Effect By Regulating The MiR-143-3p/HK2 Axis In Malignant Transformation Human Bronchial Epithelial Cells Induced By Radon

ObjectiveThe aim of the study was to explore the characterization of changes in energy metabolism during malignant transformation of human bronchial epithelial cells caused by radon exposure,then to preliminarily explore the mechanism of lncRNA PVT1 regulating Warburg effect through miR-143-3P/HK2 in the malignant transformation of BEAS-2B cells caused by long-term radon exposure.Methods1.To establish the malignant transformed BEAS-2B cells model induced by radon.BEAS-2B cells were exposed to radon for 20 min at a concentration of 20000 Bq/m3,the cells were cultured in Transwell culture dish for 3-4 days,exposed again at the same concentration and duration.It was repeated 25 times until malignant transformation occurred.Meanwhile,BEAS-2B cells,a passaged control group without radon exposure,were established,During the culturing process,measured changes in transformed phenotypes including cell proliferation,cell migration and invasion every 5 generations.2.Detected the alterations in energy metabolism and glycolytic rate-limiting enzyme in different stages(15,20,25 generations)of malignant transformation of BEAS-2B cells caused by radon exposure.glucose consumption,lactic acid,lactate dehydrogenase(LDH)activity were measured by biochemical methods.lactate dehydrogenase A(LDHA),glucose transporter 1(GLUT1),hexokinase 2(HK2),and M2 pyruvate kinase(PKM2)were detected protein expression levels by Western blot.3.The online bioprediction software starBase was used to predict and screen candidate miRNA with binding sites with lncRNA PVT1.RT-qPCR was carried out to detect the expression of lncRNA PVT1 with miR-143-3p in different stages of malignant transformation of BEAS-2B cells caused by radon exposure.RT-qPCR was used to measure the effect of lncRNA PVT1 on miR-143-3p regulation.Dual luciferase reporter assay was used to verify the possibility of targeting lncRNA PVT1 to miR-143-3p.4.The expression of miR-143-3p and HK2 genes afier radon exposure to 25-generation cell(Rn-25)was observed by transfection of si-PVT1.Cells were co-transfected with si-PVT1 and miR-143-3p inhibitor to Rn-25 cells,and the effect of lncRNA PVT1/miR-143-3P/HK2 signaling pathway on the Warburg effect after malignant transformation of BEAS-2B cells induced by long-term radon exposure was observed,and the effect of this pathway on the malignant phenotype of cells(proliferative activity,cell invasion and migration capacity)was observed.Results1.After long-term radon exposure,it was found that the growth rate of BEAS-2B cells gradually accelerated with the increase of the number of contaminated generations,and the invasive and migration rate of BEAS-2B cells was significantly enhanced from the 20th generation onwards.2.Changes in energy metabolism of BEAS-2B cells at different stages of malignant transformation caused by long-term radon exposure were found:compared with the passaged control group,glucose consumption,lactic acid and LDH content in the 20th generation of BEAS-2B-Rn were significantly increased,and there were no significant changes in LDHA,GLUT1,HK2 and PKM2 protein expression in BEAS-2B-Rn cells at the beginning of radon exposure,and from the 20th generation The expression of LDHA,GLUT1,HK2 and PKM2 did not change significantly at the beginning of radon exposure,but increased from the 20th generation when the malignant transformation phenotype appeared.It was suggested that the mode of energy metabolism of BEAS-2B cells changed from oxidative phosphorylation to aerobic glycolysis after radon exposure.3.The role of lncRNA PVT1 in regulating aerobic glycolysis in the process of malignant transformation of BEAS-2B cells through miR-143-3P/HK2:With the extension of radon exposure time,lncRNA PVT1 expression increased significantly,and cellular energy metabolism was regulated by lncRNA PVT 1/miR-143-3p/HK2,which in turn affected the malignant phenotype of cells.Compared with the passage control group,the expression of lncRNAPVT1 and HK2 increased significantly from the 20th passage,and the expression of miR-143-3p decreased significantly.After transfection of si-PVT1,aerobic glycolysis of Rn-25 cells was inhibited,and the malignant phenotype of cells decreased.The luciferase activity of miR-143-3p mimics and PVT1-MUT was not significantly reduced compared with the other groups.4.After transfection of si-PVT1,miR-143-3p of Rn-25 cells increased,and the expression of HK2 decreased significantly.The cell proliferation,number of cell clones and migration ability of si-PVT1 and miR-143-3p inhibitor were significantly enhanced compared with those in the si-PVT1 group alone.Coclusion1.Long-term radon exposure induced a shift in cellular energy supply from oxidative phosphorylation to aerobic glycolysis dominated in the malignant transformation of BEAS-2B-Rn cells.2.Long-term radon exposure induced high expression of lncRNA PVT1,promoted the expression of HK2 by inhibiting miR-143-3p,and showed Warburg effect in cellular energy metabolism,which in turn promoted the malignant transformation of BEAS-2B cells.