Molecular Mechanism Of Lactate-induced Mitochondrial Autophagy In Lung Cancer Cells Through Beclin1

Objective: Metabolic adaptability is crucial for tumor progression and when cancer cells preferentially use lactate to glucose during the Warburg effect,it mimics the occurrence of glucose starvation and induces autophagy.However,it is still unclear whether lactate plays a role in the induction of autophagy.Therefore,this article mainly discussed the role of lactate in autophagy and its molecular mechanism.Methods: Human lung cancer cells A549,H1299 and H1688 were selected as research objects.(1)Western blot was used to investigate the expressions of beclin1,p62 and LC3;(2)3-MA and HCQ were uesd to explore whether lactate-induced autophagy could be reversed;(3)Autophagosome formation was analyzed by immunofluorescence;(4)Cells were transfected with beclin1 cDNA or beclin1 siRNA and then treated different concentrations lactate to detect the expression of beclin1;(5)Dual luciferase reporter gene experiment and CHX half-life experiment were used to investigate whether lactate regulates beclin1 in transcription level or in posttranscription level;(6)Transfection of E3 ubiquitin ligase NEDD4 and CO-IP experiments were to detect the expression of beclin1 protein;(7)Cells were treated with FCCP and lactate together and then detected the expression of parkin,beclin1,and LC3 by western blot;(8)Cells were transfected by beclin1 cDNA and beclin1 siRNA,then we extracted of mitochondrial proteins and detected parkin protein expression by Western blot,cells were transfected by beclin1 cDNA and beclin1 siRNA,and we observed parkin translocation by immunofluorescence co-localization experiments;(9)MTT experiments and plate cloning experiments were to explore the biological functions of beclin1.Results:(1)After treatment with different concentrations lactate in human lung cancer cells A549,H1299 and H1688,we found that the expression levels of autophagy-related proteins beclin1 and LC3 were significantly increased,and the expression of p62 protein was significantly decreased;(2)Treated with autophagy inhibitor 3-MA and the expressions of beclin1 and LC3 proteins were decreased.Treated with autophagy inhibitor HCQ and the expressions of p62 and LC3 were accumulated,it can be inferred that autophagy was impaired.(3)The results of immunofluorescence experiments showed that red spots under the treatment of lactate were increasing while green spots were reducing;(4)In human lung cancer cells A549,H1299,and H1688,the expression of LC3 was increased and the expression of p62 was decreased after transfection of beclin1 cDNA;The expression of LC3 was decreased while the expression of p62 was increased after transfection of beclin1 siRNA;(5)The results of the dual luciferase reporter gene showed that in A549,H1299,and H1688,there was no significant change in the beclin1 promoter activity after lactate treatment;The degradation time of beclin1 was reduced from the original 6 hours to 12 hours;(6)After transfection of E3 ubiquitin ligase NEDD4 cDNA,it was found that NEDD4 promoted the degradation of beclin1;CO-IP experimental showed that under the transfection of NEDD4,the ubiquitination level of beclin1 was increased,and the ubiquitination was inhibited afterlactate treatment;(7)After the combination with lactate and FCCP,the expression of parkin,beclin1,and LC3 were increased;(8)Mitochondrial and cytoplasmic proteins were extracted after transfection with beclin1 cDNA,and it was found that beclin1 significantly promoted the translocation of parkin,and transfecting with beclin1 siRNA significantly inhibited the translocation of parkin;immunofluorescence colocalization was also obtained similar results;(9)MTT experiments showed that overexpression of beclin1 promoted cell growth,and knockdown of beclin1 inhibited cell growth;plate colony formation experiments showed that overexpression of beclin1 promoted colony formation in cells and knocked down beclin1 inhibits colony formation in cells.Conclusion: Our project demonstrated that lactate,which is a product of aerobic glycolysis,may protect cells by promoting cell mitochondrial autophagy and promoting tumorigenesis and development,and providing a clinical basis for targeted therapy of lactate and autophagy.