Effects Of MiR-146a-5p Targeting ATG7 On Autophagy,Apoptosis And Glycolysis Of Glioma Cells

BackgroundGliomas are the most common primary malignant tumors of the central nervous system,their incidence accounts for approximately 80% of primary brain tumors,and the median survival time is approximately 14～15 months.Due to the difficulty of complete resection and low cure rate,adjuvant chemotherapy and radiotherapy are often needed.However,the use of chemotherapy and radiotherapy further leads to drug resistance in tumor cells,resulting in unsatisfactory overall therapeutic effects.ATG7(autophagy related protein 7,ATG7)is a key autophagy-promoting gene that plays a critical role in the regulation of cell death and survival of various cell types.In recent years,many literatures had reported that autophagy abnormalities exist in human gliomas and played an important role in all stages of tumorigenesis and development.As a tumor suppressor,miR-146a-5p is low expressed in glioma tissues and cells,and is related to the proliferation of tumor cells.In addition,it can inhibit inflammatory response,and there is mutual regulation between inflammation and autophagy.Therefore,whether there is a relationship between them is still unknown.Therefore,demonstration the role of the two in the occurrence and development of glioma will provide theoretical or data support for its precise treatment.Objectives1.To identify whether ATG7 is a potential target of miR-146a-5p.2.To investigate the regulatory role of miR-146a-5p targeting ATG7 on autophagy,apoptosis and glycolysis of glioma cells.MethodsThe relative expression of miR-146a-5p and ATG7 were detected by real-time fluorescence quantitative PCR in normal cells(h CMEC/D3)and three kinds of glioma cells(U251,SHG-44 and U118MG).Then,transfected miR-NC group acted as controls,miR-146a-5p mimics and inhibitor were transfected into U251 and SHG-44 glioma cells respectively.After 48 hours,the expression of molecular markers related autophagy ATG5,Beclin1 LC3(microtubule-associated proteins 3,LC3),anti-apoptosis protein BCL2(B-cell lymphoma 2,Bcl2),glycolysis rate-limiting enzyme HK2(hexokinase type II,HK2),apoptosis and extracellular acidification rate were detected by western blotting,laser scanning confocal microscope,flow cytometry and Seahorse XFe bioenergetic analyzer.Results1.Compared to the normal cells(h CMEC/D3),the expression of miR-146a-5p was significantly lower in glioma cells(P<0.01).Among the three glioma cell lines,the relative expression of miR-146a-5p was the lowest in SHG-44,and higher in U251,while the expression of ATG7 was negatively correlated between.2.Compared to the NC group,the expression of ATG7 was significantly decreased in the transfected miR-146a-5p mimics group,while its significantly increased in transfected inhibitor group,both of which were statistically significant.Meanwhile,mimic and psi CHECK-ATG7-promoter were co-transfected into HEK-293,the relative fluorescence activity was lower compare with NC group.3.Compared to the NC group,the expression of autophagy related proteins ATG7,Beclin1,ATG5 and LC3 B II and autophagosome formation were significantly inhibited in the transfected mimic group,but significantly increased in the transfected inhibitor group.4.Compared to the NC group,the expression of anti-apoptosis protein BCL2 was decreased significantly in the transfected mimic group,and the proportion of cell apoptosis increased significantly,which was statistically significant.The transfection of inhibitor group was the opposite.5.Compared to the NC group,the expression of glycolysis rate-limiting enzyme HK2 were increased significantly in the transfected mimic group,and the glycolytic activity was also significantly improved,while significantly decreased in the transfection of inhibitor group.Conclusion1.In glioma cells,ATG7 is a potential target of miR-146a-5p2.miR-146a-5p can reduce the autophagic flux,and increase the apoptosis and glycolytic activity of glioma cells.